Soft anticholinergic zwitterions

ABSTRACT

Soft anticholinergic zwitterions of the formulas: 
                         
wherein R 1  and R 2  are both phenyl or one of R 1  and R 2  is phenyl and the other is cyclopentyl; and wherein each asterisk marks a chiral center; said compound having the R, S or RS stereoisomeric configuration at each chiral center unless specified otherwise, or being a mixture thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent ApplicationNo. 60/735,206, filed Nov. 10, 2005, incorporated by reference herein inits entirety and relied upon. This application is also related to U.S.application Ser. No. 11/598,079 concurrently filed herewith by thepresent inventor and claiming benefit of U.S. Provisional ApplicationNo. 60/735,207, filed Nov. 10, 2006, both incorporated by referenceherein in their entireties and relied upon.

BACKGROUND

Various anticholinergic compounds have been previously described but arenot optimal.

Muscarinic receptor antagonists are frequently used therapeutic agentsthat inhibit the effects of acetylcholine by blocking its binding tomuscarinic cholinergic receptors at neuroeffector sites on smoothmuscle, cardiac muscle, and gland cells as well as in peripherialganglia and in the central nervous system (CNS). However, their sideeffects, which can include dry mouth, photophobia, blurred vision,urinary hesitancy and retention, decreased sweating, drowsiness,dizziness, restlessness, irritability, disorientation, hallucinations,tachycardia and cardiac arrhythmias, nausea, constipation, and severeallergic reactions, often limit their clinical use, and even topicalanticholinergics can cause the same unwanted side effects.Glycopyrrolate and triotropium are among the quaternary ammoniumanticholinergics, which have reduced CNS-related side effects as theycannot cross the blood-brain barrier; however, because glycopyrrolate(or, presumably, tiotropium) is eliminated mainly as unchanged drug oractive metabolite in the urine, its administration is problematic inyoung or elderly patients and especially in uraemic patients. Toincrease the therapeutic index of anticholinergics, the soft drugapproach has been applied in a number of different designs starting fromvarious lead compounds over the past 20 years, but there is a need foryet other new soft anticholinergics. These novel muscarinic antagonists,just as all other soft drugs, are designed to elicit their intendedpharmacological effect at the site of application, but they do not needto be further metabolized upon entering the systemic circulation andthey are rapidly eliminated from the body, resulting in reduced systemicside effects and increased therapeutic index.

SUMMARY

New soft anticholinergic agents, pharmaceutical compositions containingthem, processes for their preparation and methods for eliciting ananticholinergic response, especially for treating an inflammatory orobstructive disease of the respiratory tract or for treating overactivebladder, are provided.

In one exemplary embodiment, there is provided a compound having theformula

wherein R₁ and R₂ are both phenyl or one of R₁ and R₂ is phenyl and theother is cyclopentyl; and wherein each asterisk marks a chiral center;said compound having the R, S or RS stereoisomeric configuration at eachchiral center unless otherwise specified, or being a mixture thereof.

In another exemplary embodiment there is provided a compound of theformula

In another exemplary embodiment, there is provided a compound having theformula

In other exemplary embodiments, processes for preparing the compoundsare provided.

In other exemplary embodiments, there are provided pharmaceuticalcompositions comprising one or more of the compounds of the foregoingformulas and pharmaceutically acceptable carriers therefor;pharmaceutical combinations comprising one or more of the compounds ofthe foregoing formulas and an anti-inflammatory corticosteroid, abetamimetic agent or an antiallergic agent; and methods of using thesubject compositions and combinations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing mydriatic response (change in pupil size) withtime after topical administration of zwitterion Compound (cc), itsparent soft drug ester Compound (d) or glycopyrrolate in rabbits.

FIG. 2 is a graph showing mydriatic response (change in pupil size) withtime after intravenous administration of 2.5 μmol/kg of Compound (cc) orglycopyrrolate in rabbits (n=4).

FIG. 3 is a graph showing the effect of Compound (cc) (5 μmol/kg) on theresting heart rate in anesthetized rats (n=4) as compared to controlrats.

FIG. 4 is a graph showing the protective effect of varying doses ofCompound (cc) (0.25, 0.5, 1.25, 2.5 and 5.0 μmol/kg) and glycopyrrolate(0.5 μmol/kg) on carbachol-induced bradycardia (n=4), where theasterisks indicate p<0.005 compared to glycopyrrolate.

FIG. 5 is a graph depicting a mean plasma concentration-time profileafter intravenous injection of Compound (cc) at a dose of 30 mg/kg inrats (n=4), where the line represents data predicted by the twocompartment model (Table 3).

FIG. 6 is a graph of mydriatic activities of various zwitterionicisomers at 0.1% concentrations over a seven hour period.

FIG. 7 is a graph comparing the mydriatic activity of the most activezwitterionic isomers with glycopyrrolate at 0.1% concentrations.

FIG. 8 is a graph showing the time course of action of differentanticholinergics, including Compounds (w) and (aa), on electricallystimulated guinea pig trachea.

FIG. 9 is a graph showing the time course of the effect of differentanticholingerics including Compounds (w) and (aa), after wash out of thetest drug on electrically stimulated guinea pig trachea.

DETAILED DESCRIPTION

Throughout this specification, the following definitions, generalstatements and illustrations are applicable:

The patents, published applications, and scientific literature referredto herein establish the knowledge of those with skill in the art and arehereby incorporated by reference in their entirety to the same extent asif each was specifically and individually indicated to be incorporatedby reference. Any conflict between any reference cited herein and thespecific teachings of this specification shall be resolved in favor ofthe latter. Likewise, any conflict between an art-understood definitionof a word or phrase and a definition of the word or phrase asspecifically taught in this specification shall be resolved in favor ofthe latter.

As used herein, whether in a transitional phrase or in the body of aclaim, the terms “comprise(s)” and “comprising” are to be interpreted ashaving an open-ended meaning. That is, the terms are to be interpretedsynonymously with the phrases “having at least” or “including at least”.When used in the context of a process, the term “comprising” means thatthe process includes at least the recited steps, but may includeadditional steps. When used in the context of a composition, the term“comprising” means that the composition includes at least the recitedfeatures or components, but may also include additional features orcomponents.

The terms “consists essentially of” or “consisting essentially of” havea partially closed meaning, that is, they do not permit inclusion ofsteps or features or components which would substantially change theessential characteristics of a process or composition; for example,steps or features or components which would significantly interfere withthe desired properties of the compounds or compositions describedherein, i.e., the process or composition is limited to the specifiedsteps or materials and those which do not materially affect its basicand novel characteristics. The basic and novel features herein are theprovision of compounds of formula (Ia) and (Ib) and combinations ofthose compounds with other drugs, particularly with antiinflammatorysteroids, especially loteprednol etabonate or etiprednoldichloroacetate, and most especially in the case of loteprenol etabonate(LE) further including an inactive metabolite enhancing agent for the LEas further defined hereinafter.

The terms “consists of” and “consists” are closed terminology and allowonly for the inclusion of the recited steps or features or components.

As used herein, the singular forms “a,” “an” and “the” specifically alsoencompass the plural forms of the terms to which they refer, unless thecontent clearly dictates otherwise.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” or “approximately” is used herein to modify a numerical valueabove and below the stated value by a variance of 20%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable can be equal to any integer valueof the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variablecan be equal to any real value of the numerical range, including theend-points of the range. As an example, a variable which is described ashaving values between 0 and 2, can be 0, 1 or 2 for variables which areinherently discrete, and can be 0.0, 0.1, 0.01, 0.001, or any other realvalue for variables which are inherently continuous.

In the specification and claims, the singular forms include pluralreferents unless the context clearly dictates otherwise. As used herein,unless specifically indicated otherwise, the word “or” is used in the“inclusive” sense of “and/or” and not the “exclusive” sense of“either/or.”

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present inventionpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skill in the art. Standardreference works setting forth the general principles of pharmacologyinclude Goodman and Gilman's The Pharmacological Basis of Therapeutics,10^(th) Ed., McGraw Hill Companies Inc., New York (2001).

As used herein, “treating” means reducing, preventing, hindering orinhibiting the development of, controlling, alleviating and/or reversingthe symptoms in the individual to which a combination or composition asdescribed herein has been administered, as compared to the symptoms ofan individual not being treated as described herein. A practitioner willappreciate that the combinations, compositions, dosage forms and methodsdescribed herein are to be used in concomitance with continuous clinicalevaluations by a skilled practitioner (physician or veterinarian) todetermine subsequent therapy. Such evaluation will aid and inform inevaluating whether to increase, reduce or continue a particulartreatment dose, and/or to alter the mode of administration.

The methods described herein are intended for use with anysubject/patient that may experience their benefits. Thus, in accordanceherewith, the terms “subjects” as well as “patients,” “individuals” and“warm-blooded animals” include humans as well as non-human subjects,particularly domesticated animals, particularly dogs, cats, horses andcows, as well as other farm animals, zoo animals and/or endangeredspecies.

The compound of formula (Ib), which is of particular interest, can benamed 6β,7β-epoxy-3-hydroxy-8-carboxymethyl-8-methyl-1αH,5αH-tropanium,di-2-thienylglycolate inner salt and is also referred to herein asCompound (aa).

In formula (Ia), the compounds having the R configuration with respectto chiral center 2 are of particular interest.

In the compounds of formula (Ia), compounds wherein one of R₁ and R₂ isphenyl and the other is cyclopentyl are of particular interest.

Also of particular interest are the compounds of the formula:

The following specific compounds of formula (Ia) are of particularinterest:

-   -   (bb) (±)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt;    -   (cc) (2R)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt;    -   (dd) (2R,1′R, 3′R)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt;    -   (ee) (2R,1′S, 3′R)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt;    -   (ff) (2R,1′R, 3′S)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt;    -   (gg) (2R,1′S, 3′S)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt;    -   (hh) (2S,1′R, 3′R)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt;    -   (ii) (2S,1′S, 3′R)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt;    -   (jj) (2S,1′R, 3′S)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt; and    -   (kk) (2S,1′S, 3′S)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt.

Of these, particular mention may be made of:

-   -   (bb) (±)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt; and    -   (cc) (2R)        3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidinium        inner salt.

Various methods of making the instant compounds are illustratedhereinafter. Generally speaking, the compounds of formula (Ia) can beprepared by hydrolysis of the corresponding esters of the formula

wherein R₁ and R₂, the asterisks and the stereoisomeric configurationsare as defined above; R is C₁-C₈ alkyl, straight or branched chain; andX⁻ is an anion with a single negative charge. The compounds of formula(IIa) are novel anticholinergic esters described and claimed in U.S.application Ser. No. 11/598,079, concurrently filed herewith by thepresent inventor and incorporated by reference herein in its entiretyand relied upon. The compounds of formula (IIa) can be prepared byreacting a bromoacetate of the formulaBrCH₂COORwherein R is as defined above, with a compound of the formula

wherein R₁ and R₂, the asterisks and the stereoisomeric configurationsare as defined above, and optionally separating the individualstereoisomers to afford a compound of formula (IIa) and, when desired,exchanging the bromine anion with a different X⁻ anion wherein X⁻ is asdefined above but other than Br⁻.

In a particular embodiment, the compound of formula (IIIa) has the Rconfiguration with respect to chiral center 2.

In another particular embodiment, the compound of formula (IIIa) has theconfiguration R or S with respect to chiral center 1′. The compound offormula (IIa) can also be made stereospecifically with respect to chiralcenter 3′.

In another embodiment, the process includes separating the individualstereoisomers of the compound of formula (Ia) after their formation tothe extent possible.

In one particular embodiment, the process comprises preparing a compoundof formula (Ia-i) or (Ia-ii) by hydrolyzing the corresponding methylester in aqueous sodium hydroxide solution.

In analogous fashion, methods of making the compound of formula (Ib) areillustrated hereinafter. Generally speaking, two alternate routes areproposed. One route comprises hydrolysis (for example, acid hydrolysis)of the corresponding esters of the formula

wherein the asterisks, stereoisomeric configuration, R and X⁻ are asdefined above. The compounds of formula (IIb) are novel anticholinergicesters described and claimed in the U.S. application Ser. No.11/598,079, concurrently filed herewith by the present inventor andincorporated by reference herein in its entirety and relied upon. Thecompounds of formula (IIb) can be prepared by reacting a bromoacetate ofthe formula:BrCH₂COORwherein R is as defined above, with a compound of the formula

and optionally separating the individual stereoisomers to afford acompound of formula (IIb) and, when desired, exchanging the bromineanion with a different X⁻ anion wherein X⁻ is as defined above but otherthan Br⁻.

In an alternative route to the compound of formula (Ib), a scopine esterof the formula

is reacted with trichloroethyl bromoacetate to afford the trichloroethylester

which is subjected to acid hydrolysis, using zinc dust and acetic acid,to afford the compound of formula (Ib).

The compounds of formulas (Ia) and (Ib) are of use as pharmaceuticalagents because of their anticholinergic activity. An anticholinergicallyeffective amount of such an agent inhibits the effect of acetycholine byblocking its binding to muscarinic cholinergic receptors atneuroeffector sites. Subjects in need of a method of eliciting ananticholinergic response are those suffering from conditions whichrespond to treatment with an anticholinergic agent. Such conditionsinclude obstructive diseases of the respiratory tract, for exampleasthma and chronic obstructive pulmonary disease, vagally induced sinusbradycardia and heart rhythm disorders, spasms, for example in thegastrointestinal tract or urinary tract (including overactive bladder)and in menstrual disorders. The compounds of formulas (Ia) and (Ib) canalso be used to induce short-acting mydriasis and thus can be used todilate the pupils of the eyes in vision testing. Other uses of thecompounds of formulas (Ia) and (Ib) include the treatment of ulcers aswell as topical use as an antiperspirant in the treatment hyperhydrosis(sweating).

The compounds of formula (Ia) and (Ib) are particularly useful in thetreatment of obstructive diseases of the respiratory tract. Theexpression “obstructive disease of the respiratory tract” includesbreathing disorders such as asthma, bronchitis, chronic obstructivepulmonary disease (COPD), allergic rhinitis and infectious rhinitis.

“Asthma” refers to a chronic lung disease causing bronchoconstriction(narrowing of the airways) due to inflammation (swelling) and tighteningof the muscles around the airways. The inflammation also causes anincrease in mucus production, which causes coughing that may continuefor extended periods. Asthma is generally characterized by recurrentepisodes of breathlessness, wheezing, coughing, and chest tightness,termed exacerbations. The severity of exacerbations can range from mildto life threatening. The exacerbations can be a result of exposure toe.g. respiratory infections, dust, mold, pollen, cold air, exercise,stress, tobacco smoke, and air pollutants.

“COPD” refers to chronic obstructive pulmonary disease, primarily butnot necessarily associated with past and present cigarette smoking. Itinvolves airflow obstruction, mainly associated with emphysema andchronic bronchitis. Emphysema causes irreversible lung damage byweakening and breaking the air sacs within the lungs. Chronic bronchitisis an inflammatory disease, which increases mucus in the airways andbacterial infections in the bronchial tubes, resulting in obstructedairflow.

“Allergic rhinitis” refers to acute rhinitis or nasal rhinitis,including hay fever. It is caused by allergens such as pollen or dust.It may produce sneezing, congestion, runny nose, and itchiness in thenose, throat, eyes, and ears.

“Infectious rhinitis” refers to acute rhinitis or nasal rhinitis ofinfectious origin. It is caused by upper respiratory tract infection byinfectious rhinoviruses, coronaviruses, influenza viruses, parainfluenzaviruses, respiratory syncytical virus, adenoviruses, coxsackieviruses,echoviruses, or Group A beta-hemolytic Streptococci and is genericallyreferred to as the common cold. It may produce sneezing, congestion,runny nose, and itchiness in the nose, throat, eyes, and ears.

The compounds of formula (Ia) and (Ib) are also particularly useful inthe treatment of overactive bladder (OAB).

Overactive bladder is a treatable medical condition that is estimated toaffect 17 to 20 million people in the United States. Symptoms ofoveractive bladder can include urinary frequency, urinary urgency,urinary urge incontinence (accidental loss of urine) due to a sudden andunstoppable need to urinate, nocturia (the disturbance of nighttimesleep because of the need to urinate) or enuresis resulting fromoveractivity of the detrusor muscle (the smooth muscle of the bladderwhich contracts and causes it to empty).

Neurogenic overactive bladder (or neurogenic bladder) is a type ofoveractive bladder which occurs as a result of detrusor muscleoveractivity referred to as detrusor hyperreflexia, secondary to knownneurologic disorders. Patients with neurologic disorders, such asstroke, Parkinson's disease, diabetes, multiple sclerosis, peripheralneuropathy, or spinal cord lesions often suffer from neurogenicoveractive bladder. In contrast, non-neurogenic overactive bladderoccurs as a result of detrusor muscle overactivity referred to asdetrusor muscle instability. Detrusor muscle instability can arise fromnon-neurological abnormalities, such as bladder stones, muscle disease,urinary tract infection or drug side effects or can be idiopathic.

Due to the enormous complexity of micturition (the act of urination), anexact mechanism which causes overactive bladder is not known. Overactivebladder can result from hypersensitivity of sensory neurons of theurinary bladder, arising from various factors including inflammatoryconditions, hormonal imbalances, and prostate hypertrophy. Destructionof the sensory nerve fibers, either from a crushing injury to the sacralregion of the spinal cord, or from a disease that causes damage to thedorsal root fibers as they enter the spinal cord can also lead tooveractive bladder. In addition, damage to the spinal cord or brain stemcausing interruption of transmitted signals can lead to abnormalities inmicturition. Therefore, both peripheral and central mechanisms can beinvolved in mediating the altered activity in overactive bladder.

Current treatments for overactive bladder include medication, dietmodification, programs in bladder training, electrical stimulation, andsurgery. Currently, antimuscarinics (which are members of the generalclass of anticholinergics) are the primary medication used for thetreatment of overactive bladder. The antimuscarinic, oxybutynin, hasbeen the mainstay of treatment for overactive bladder. However,treatment with known antimuscarinics suffers from limited efficacy andside effects such as dry mouth, dry eyes, dry vagina, blurred vision,cardiac side effects, such as palpitations and arrhythmia, drowsiness,urinary retention, weight gain, hypertension and constipation, whichhave proven difficult for some individuals to tolerate. Thus, the needfor new anticholinergic agents is evident.

The compounds of formulas (Ia) and (Ib) are the zwitterion metabolitesof the corresponding esters of formulas (IIa) and (IIb). While thecompounds of formulas (Ia) and (Ib) are less active than thecorresponding esters (by about an order of magnitude), the zwitterionsare very rapidly eliminated from the systemic circulation mainly throughurinary excretion in their unchanged form. This makes them particularlydesirable for use in treating urinary tract disorders, especiallyoveractive bladder. Moreover, their M₃/M₂ subtype selectively is greatlyenhanced as compared to the parent esters, reducing the likelihood ofcardiac side effects. The significantly reduced toxicity of thezwitterions also makes the zwitterions particularly desirable forlong-term use, for example in the treatment of chronic conditions suchas COPD or asthma.

The compounds of formula (Ia) or (Ib) may be used on their own orcombined with other active substances of formula (Ia) or (Ib) accordingto the invention.

The compounds of formula (Ia) or (Ib) may optionally also be combinedwith other pharmacologically active substances. These include, inparticular, betamimetics, antiallergic agents, and corticosteroids (alsotermed “anti-inflammatory steroids”, “anti-inflammatory costicosteroids”or simply “steroids”) and combinations of these active substances. Thecombinations with betaminetics, antiallergics or corticosteroids are ofinterest in the treatment of obstructive diseases of the respiratorytract, especially COPD or asthma. Accordingly, they are intended foradministration by oral inhalation as powders or aerosols.

Examples of betamimetics which may be used in conjunction with thecompounds of formula (Ia) or (Ib) include compounds selected from thegroup consisting of bambuterol, bitolterol, carbuterol, clenbuterol,fenoterol, formoterol, hexoprenaline, ibuterol, pirbuterol, procaterol,reproterol, salmeterol, sulfphonterol, terbutaline, tulobuterol,4-hydroxy-7-[2-{[2-{[3-(2-phenylethoxy)propyl]sulfonyl}ethyl]amino}ethyl]-2(3H)-benzothiazolone,1-(2-fluoro-4-hydroxyphenyl)-2-[4-(1-benzimidazolyl)-2-methyl-2-butylamino]ethanol,1-[3-(4-methoxybenzylamino)-4-hydroxyphenyl]-2-[4-(1-benzimidazolyl)-2-methyl-2-butylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-N,N-dimethylaminophenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-methoxyphenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-n-butyloxyphenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-{4-[3-(4-methoxyphenyl)-1,2,4-triazol-3-yl]-2-methyl-2-butylamino}ethanol,5-hydroxy-8-(1-hydroxy-2-isopropylaminobutyl)-2H-1,4-benzoxazin-3-(4H)-one,1-(4-amino-3-chloro-5-trifluoromethylphenyl)-2-tert.-butylamino)ethanoland1-(4-ethoxycarbonylamino-3-cyano-5-fluorophenyl)-2-(tert.-butylamino)ethanol,optionally in the form of their racemates, their enantiomers, theirdiastereomers, as well as optionally their pharmacologically acceptableacid addition salts and hydrates. It is particularly preferable to use,as betamimetics, active substances of this kind, combined with thecompounds of formula (Ia) or (Ib), selected from among fenoterol,formoterol, salmeterol,1-[3-(4-methoxybenzylamino)-4-hydroxyphenyl]-2-[4-(1benzimidazolyl)-2-methyl-2-butylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-N,N-dimethylaminophenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-methoxyphenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-n-butyloxyphenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-{4-[3-(4-methoxyphenyl)-1,2,4-triazol-3-yl]-2-methyl-2-butylamino}ethanol,optionally in the form of their racemates, their enantiomers, theirdiastereomers, as well as optionally their pharmacologically acceptableacid addition salts and hydrates. Of the betamimetics mentioned above,the compounds formoterol and salmeterol, optionally in the form of theirracemates, their enantiomers, their diastereomers, as well as optionallytheir pharmacologically acceptable acid addition salts and hydrates, areparticularly important.

The acid addition salts of the betamimetics selected from among thehydrochloride, hydrobromide, sulfate, phosphate, fumarate,methanesulfonate and xinafoate are preferred. In the case of salmeterol,the salts selected from among the hydrochloride, sulfate and xinafoateare particularly preferred, especially the sulfates and xinafoates. Inthe case of formoterol, the salts selected from among the hydrochloride,sulfate and fumarate are particularly preferred, especially thehydrochloride and fumarate. Of outstanding importance is formoterolfumarate.

The corticosteroids which may optionally be used in conjunction with thecompounds of formula (Ia) or (Ib), include compounds selected from amongflunisolide, beclomethasone, triamcinolone, budesonide, fluticasone,mometasone, ciclesonide, rofleponide, GW 215864, KSR 592, ST-126,loteprednol etabonate, etiprednol dichloroacetate and dexamethasone. Thepreferred corticosteroids are those selected from among flunisolide,beclomethasone, triamcinolone, loteprednol etabonate, etiprednoldichloroacetate, budesonide, fluticasone, mometasone, ciclesonide anddexamethasone, while budesonide, fluticasone, loteprednol etabonate,etiprednol dichloroacetate, mometasone and ciclesonide, especiallybudesonide, fluticasone, loteprednol etabonate and etiprednoldichloroacetate, are of particular importance. Any reference to steroidsherein also includes a reference to salts or derivatives which may beformed from the steroids. Examples of possible salts or derivativesinclude: sodium salts, sulfobenzoates, phosphates, isonicotinates,acetates, propionates, dihydrogen phosphates, palmitates, pivalates orfuroates. The corticosteroids may optionally also be in the form oftheir hydrates.

When the corticosteroid is loteprednol etabonate, it may beadvantageously combined with an enhancing agent selected from the groupconsisting of:

-   -   (a) 11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylic acid        (cortienic acid, or CA);    -   (b) 11β,17α-dihydroxyandrost-1,4-dien-3-one-17β-carboxylic acid        (Δ¹ cortienic acid or Δ¹-CA);    -   (c) methyl 11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylate        (cortienic acid methyl ester, or MeCA);    -   (d) ethyl 11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylate        (cortienic acid ethyl ester, or EtCA);    -   (e) methyl        11β,17α-dihydroxyandrost-1,4-dien-3-one-17β-carboxylate ((Δ¹        cortienic acid methyl ester, or Δ¹-MeCA); and    -   (f) ethyl        11β,17α-dihydroxyandrost-1,4-dien-3-one-17β-carboxylate (Δ¹        cortienic acid ethyl ester, or Δ¹-EtCA),        wherein the mole ratio of loteprednol etabonate to enhancing        agent is from about 5:1 to about 0.5:1. Such combinations with        these inactive metabolites are described in detail in WO        2005/000317 A1, incorporated by reference herein in its entirety        and relied upon.

Examples of antiallergic agents which may be used as a combination withthe compounds of formula (Ia) or (Ib) include epinastin, cetirizin,azelastin, fexofenadin, levocabastin, loratadine, mizolastin, ketotifen,emedastin, dimetinden, clemastine, bamipin, cexchloropheniramine,pheniramine, doxylamine, chlorophenoxamine, dimenhydrinate,diphenhydramine, promethazine, ebastin, desloratidine and meclizine.Preferred antiallergic agents which may be used in combination with thecompounds of formula (Ia) or (Ib) are selected from among epinastin,cetirizin, azelastin, fexofenadin, levocabastin, loratadine, ebastin,desloratidine and mizolastin, epinastin and desloratidine beingparticularly preferred. Any reference to the abovementioned antiallergicagents also includes a reference to any pharmacologically acceptableacid addition salts thereof which may exist.

When the compounds of formula (Ia) or (Ib) are used in conjunction withother active substances, the combination with steroids or betamimeticsis particularly preferred of the various categories of compoundsmentioned above.

Whether or not the compounds of formula (Ia) or (Ib) are used inconjunction with other active substances as described above, they aretypically administered in the form of a pharmaceutical compositioncomprising an anticholinergically effective amount of a compound offormula (Ia) or (Ib) and a non-toxic pharmaceutically acceptable carriertherefor. Pharmaceutically acceptable carriers, or diluents, arewell-known in the art. The carriers may be any inert material, organicor inorganic, suitable for administration, such as: water, gelatin, gumarabic, lactose, microcrystalline cellulose, starch, sodium starchglycolate, calcium hydrogen phosphate, magnesium stearate, talcum,colloidal silicon dioxide, and the like. Such compositions may alsocontain other pharmaceutically active agents, as noted above, and/orconventional additives such as stabilizers, wetting agents, emulsifiers,flavoring agents, buffers, binders, disintegrants, lubricants, glidants,antiadherents, propellants, and the like. The carrier, e.g., non-activeingredient, can be just (sterile) water with the pH adjusted to wherethe active pharmaceutical agent is very soluble. It is preferred thatthe pH be at or near 7. Alternatively and preferably, the non-activecarrier agent should be physiological saline with the pH adjustedappropriately.

The novel compounds of formula (Ia) or (Ib) can be administered in anysuitable way. The compounds can be made up in solid or liquid form, suchas tablets, capsules, powders, syrups, elixirs and the like, aerosols,sterile solutions, suspensions or emulsions, and the like.

The compounds of formula (Ia) or (Ib) can be brought into suitabledosage forms, such as compositions for administration through the oral,rectal, trandermal, parenteral, nasal, pulmonary (typically via oralinhalation) or topical (including ophthalmic) route in accordance withaccepted pharmaceutical procedures. The route of administration and thusthe dosage form will be chosen in light of the condition to be treatedwith the instant anticholinergic agents. By way of illustration only,when the compound of formula (Ia) or (Ib) is administered to treat COPDor asthma, of other serious obstructive disease of the respiratorytract, the compounds may be advantageously administered via inhalationor insufflation; for such purposes, the compounds are advantageously inthe form of an aerosol or a powder for inhalation. When administered totreat less serious respiratory disorders such as rhinitis, a nasalspray, mist or gel may be advantageous. For inducing mydriasis, anophthalmic formulation such as eye drops may be most appropriate. ForOAB, a formulation for oral administration such as tablet or capsules ora transdermal preparation may be preferred. For treating hyperhydrosis,an topical preparation formulated as an antiperspirant stick, gel,spray, cream or the like would be preferred.

For purposes of illustration, dosages are expressed based on theinhalation of an aerosol solution, such as the product AtroventInhalation Aerosol (Boehringer Ingelheim). Adjustments in dosages foradministration by other modes of inhaled administration are well knownto those skilled in the art.

In general, a therapeutically effective amount of compound of formula(Ia) or (Ib) is from about 4 μg to about 1,000 μg, e.g., from about 30μg to about 1,000 μg or from about 200 μg to about 1000 μg. However, theexact dosage of the specific compound of formula (Ia) or (Ib) will varydepending on its potency, the mode of administration, the age and weightof the subject and the severity of the condition to be treated. Thedaily dosage may, for example, range from about 0.03 μg to about 40 μgper kg of body weight, administered singly or multiply in doses e.g.from about 3 μg to about 4,000 μg each. The compounds of formula (Ia) or(Ib) can be administered from one to four times daily, e.g., once ortwice daily.

The dosage form for inhalation can be an aerosol. The minimum amount ofan aerosol delivery is about 0.2 ml and the maximum aerosol delivery isabout 5 ml. The concentration of the compounds of formula (Ia) or (Ib)may vary as long as the total amount of spray delivered is within theabout 0.2 to about 5 ml amount and as long as it delivers ananticholinergically effective amount of the compound of formula (Ia) or(Ib). It is well known to those skilled in the art that if theconcentration is higher, one gives a smaller dose to deliver the sameeffective amount.

The dosage form for inhalation can also be via intranasal spray. Theminimum amount of an aerosol delivery is about 0.02 ml per nostril andthe maximum aerosol delivery is about 0.2 ml per nostril. Theconcentration of the compounds of formula (Ia) or (Ib) may vary as longas the total amount of spray delivered is within about 0.02 ml pernostril to about 0.2 ml per nostril, e.g., between about 0.05 ml pernostril and about 0.08 ml per nostril, and it delivers ananticholinergically effective amount of the compound of formula (Ia) or(Ib).

Of course, the volume of aerosol or intranasal spray for delivering ananticholinergically effective amount of the compound of formula (Ia) or(Ib) depends upon the concentration of the compound in the aerosol orintranasal spray, i.e., higher concentrations of the compound of formula(Ia) or (Ib) require smaller dosage volumes to deliver a therapeuticallyeffective amount and lower concentrations of the compound of formula(Ia) or (Ib) require larger dosage volumes to deliver the sameanticholinergically effective amount.

Aerosols for inhalation of various pharmaceutical agents are well knownto those skilled in the art, including many aerosols for treatingasthma. Aerosols may be produced with a nebulizer. Typically, thenebulizer is charged with a carrier solution and the compound of formula(Ia) or (Ib) in an amount sufficient to effectively deliver ananticholinergically effective amount of the compound of formula (Ia) or(Ib). For instance, depending upon the nebulizer and its operatingconditions, the nebulizer may be charged with several hundred mg ofanticholinergic compound in order to deliver about 4 μg to about 1000μg, e.g., from about 30 μg to about 1000 μg or from about 150 μg toabout 800 μg, of the compound of formula (Ia) or (Ib).

The dosage form for inhalation may also be in powder form. Powders forinhalation of various pharmaceutical agents are well known to thoseskilled in the art, including many powders for treating asthma. When thedosage form is a powder, the compounds of formula (Ia) or (Ib) can beadministered in pure form or diluted with an inert carrier. When aninert carrier is used, the compounds are compounded such that the totalamount of powder delivered delivers an “effective amount” of thecompounds according to the invention. The actual concentration of theactive compound may vary. If the concentration is lower, then morepowder must be delivered, if the concentration is higher, less totalmaterial must be delivered to provide an effective amount of the activecompound according to the invention. Any of the foregoing pharmaceuticalcompositions may further comprise one or more additional activesubstances, particularly corticosteroids and/or betamimetics asdiscussed earlier.

“Pharmaceutically acceptable” refers to those properties and/orsubstances which are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability.

Suitable preparations for administering the compounds of formula (Ia) or(Ib) include tablets, capsules, suppositories, solutions, etc. Ofparticular importance (particularly when treating asthma or COPD orother respiratory disorders) is the administration of the compounds byinhalation. The proportion of pharmaceutically active compound orcompounds should be in the range from 0.05 to 90% by weight, preferably0.1 to 50% by weight of the total composition. Suitable tablets may beobtained, for example, by mixing the active substance(s) with knownexcipients, for example inert diluents such as calcium carbonate,calcium phosphate or lactose, disintegrants such as corn starch oralginic acid, binders such as starch or gelatin, lubricants such asmagnesium stearate or talc and/or agents for delaying release, such ascarboxymethyl cellulose, cellulose acetate phthalate, or polyvinylacetate. The tablets may also comprise several layers. Tablets and othersolid oral formulations are of particular interest in the treatment ofOAB or ulcers while ophthalmic solutions, suspensions and gels are ofspecial interest for inducing mydriasis and topical gels, solids andsprays are of particular use as antiperspirants.

Coated tablets may be prepared accordingly by coating cores producedanalogously to the tablets with substances normally used for tabletcoatings, for example collidone or shellac, gum arabic, talc, titaniumdioxide or sugar. To achieve delayed release or preventincompatibilities the core may also consist of a number of layers.Similarly the tablet coating may consist of a number or layers toachieve delayed release, possibly using the excipients mentioned abovefor the tablets.

Syrups or elixirs containing the active substances of formulas (Ia) or(Ib) or combinations thereof as described above may additionally containa sweetener such as saccharin, cyclamate, aspartame, sucralose, glycerolor sugar and a flavor enhancer, e.g. a flavoring such as vanillin ororange extract. They may also contain suspension adjuvants or thickenerssuch as sodium carboxymethyl cellulose, wetting agents such as, forexample, condensation products of fatty alcohols with ethylene oxide, orpreservatives such as p-hydroxybenzoates.

Solutions are prepared in the usual way, e.g. with the addition ofisotonic agents, preservatives such as p-hydroxybenzoates, orstabilizers such as alkali metal salts of ethylenediamine tetraaceticacid, optionally using emulsifiers and/or dispersants, while if water isused as the diluent, for example, organic solvents may optionally beused as solvating agents or dissolving aids, and transferred intoinjection vials or ampules or infusion bottles.

Capsules containing one or more active substances or combinations ofactive substances may for example be prepared by mixing the activesubstances with inert carriers such as lactose or sorbitol and packingthem into gelatin capsules. Suitable suppositories may be made forexample by mixing with carriers provided for this purpose, such asneutral fats or polyethyleneglycol or the derivatives thereof.Excipients which may be used include, for example, water,pharmaceutically acceptable organic solvents such as paraffins (e.g.petroleum fractions), vegetable oils (e.g. groundnut or sesame oil),mono- or polyfunctional alcohols (e.g. ethanol or glycerol), carrierssuch as e.g. natural mineral powders (e.g. kaolins, clays, talc, chalk),synthetic mineral powders (e.g. highly dispersed silicic acid andsilicates), sugars (e.g. cane sugar, lactose and glucose), emulsifiers(e.g. lignin, spent sulfite liquors, methylcellulose, starch andpolyvinylpyrrolidone) and lubricants (e.g. magnesium stearate, talc,stearic acid and sodium lauryl sulphate).

The preparations are administered by the usual methods, preferably byinhalation in the treatment of asthma or COPD or other respiratorydisorders. For oral administration the tablets may, of course, contain,apart from the above-mentioned carriers, additives such as sodiumcitrate, calcium carbonate and dicalcium phosphate together with variousadditives such as starch, preferably potato starch, gelatin and thelike. Moreover, lubricants such as magnesium stearate, sodium laurylsulfate and talc may be used at the same time for the tablettingprocess. In the case of aqueous suspensions the active substances may becombined with various flavor enhancers or colorings in addition to theexcipients mentioned above.

The dosage of the compounds of formula (Ia) and (Ib) is naturallygreatly dependent on the route of administration and the complaint to betreated. When administered by inhalation the compounds of formula (Ia)or (Ib) are characterized by high efficacy even at doses in the μgrange. The compounds of formula (Ia) or (Ib) can also be usedeffectively above the μg range. The dosage may then be in the gramrange, for example. Particularly when administered by a method otherthan inhalation, the compounds according to the invention may be givenin higher doses (in the range from 3 to 1000 mg, for example, althoughthis does not imply any limitation).

The compounds of formula (Ia) and (Ib), combinations of a compound offormula (Ia) or (Ib) with one or more other active agents, andcompositions comprising a compound of formula (Ia) or (Ib), with orwithout one or more other active agents, as described hereinabove arethus useful in a method for eliciting an anticholinergic response in asubject in need of same, comprising administering to said subject ananticholinergically effective amount of said compound or composition. Inparticular embodiments, the method is for treating an obstructivedisease of the respiratory tract, especially when the disease is chronicobstructive pulmonary disease or asthma, or for treating overactivebladder. In another embodiment, the method comprises inducing mydriasisin the eye(s) of a subject in need of such treatment, comprisingtopically applying to the eye(s) of said subject a mydriaticallyeffective amount of a compound of formula (Ia) or (Ib) or combination orcomposition comprising it as described hereinabove. Use of compounds offormula (Ia) or (Ib) in the preparation of a medicament for treating acondition responsive to an anticholinergic agent (such as any of theseconditions disclosed above) is likewise provided herein.

In particular embodiments there are provided combinations of thecompound of formula (Ia) or (Ib) with other active agents, especiallyone or more antiinflammatory corticosteroids, betamimetic agents orantiallergic agents. In the combination products, the active agents arepresent in a combined amount effective to treat the target condition,especially to treat an obstructive disease of the respiratory tract,most especially to treat chronic obstructive pulmonary disease orasthma. In preferred embodiments, the other active agent is abetamimetic agent or an antiinflammatory corticosteroid. Of particularinterest are combinations of a compound of formula (Ia) or (Ib) and acosticosteroid, especially loteprednol etabonate or etiprednoldichloroacetate. When loteprednol etabonate is selected as thecorticosteroid, its activity can be enhanced by combination withcortienic acid or Δ¹-cortienic acid or a methyl or ethyl ester ofcortienic acid or Δ¹-cortienic acid, in a mole ratio of from about 5:1to about 0.5:1. A molar ratio of about 1:1, which can be approximated bya 1:1 ratio by weight, is particularly convenient.

Initial Studies

Purpose

Evaluation of the zwitterionic common metabolite of a novel series ofN-substituted soft analogs of glycopyrrolate both as racemates and as 2Risomers.

Methods

Activities have been assessed using both in vitro (receptor-bindingassay, guinea pig ileum pA₂-assay) and in vivo techniques (rabbitmydriatic response, rat cardiac effects). Pharmacokineticcharacterizations in rats also have been performed.

Results

The metabolite was highly water-soluble and very stable in buffersolutions as well as in rat biological media. Following i.v.administration in rats, it was very rapidly eliminated, mainly throughrenal excretion with a half-life of about 10 min. Receptor-binding andguinea pig ileum assays indicated this metabolite as more than an orderof magnitude less active than its parent soft drugs or glycopyrrolate.Moderate M₃/M₂ muscarinic-receptor subtype-selectivity was observed,further reducing the likelihood of cardiac side-effects. The metaboliteshowed some mydriatic effect and some protecting effect againstcarbachol-induced bradycardia, but of much shorter durations thanglycopyrrolate, and it had no effect on resting heart rate.

Conclusions

N-substituted zwitterionic metabolites retain some, but reduced activityof their parent quaternary ammonium-ester soft anticholinergic drugs,and they are very rapidly eliminated from the systemic circulation.

A recently developed series of N-substituted soft glycopyrrolateanticholinergics [exemplified below and represented by formula (IIa)hereinabove] have a zwitterionic metabolite in which the positivequaternary nitrogen and the negative acid moiety formed by hydrolysisare spatially very close, and, hence, the overall electron distributionis somewhat similar to that of the neutral compound, which is active.Therefore, because this metabolite might still retain some activity, adetailed investigation of its pharmacokinetic and pharmacodynamics(PK/PD) was undertaken to ensure that the corresponding N-substitutedsoft anticholinergics still can be considered as undergoing a facile,essentially one-step metabolic deactivation as required by theprinciples of soft drug design. Because stereospecificity is known toaffect pharmacological activity at muscarinic receptors, in addition tothe racemic metabolite, (±)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, the corresponding 2R isomer has also been prepared andexamined.

Receptor binding affinity is a major determinant of drug activity. Formuscarinic receptors, five subtypes, M₁-M₅, have been found and clonedfrom human tissue, and there is sufficient correlation among thesemolecular subtypes and pharmacological subtypes to warrant use of aunified M₁-M₅ notation. Subtype selectivity (e.g., M₃/M₂) could beuseful in eliminating many potential side effects, but most currentlyused anticholinergics show no subtype selectivity; a few newer ones thatshow muscarinic receptor subtype selectivity are being pursued fordevelopment. For soft anticholinergics, such subtype selectivity couldalso further enhance their therapeutic advantage by further decreasingtheir side effects.

In the present study, chemical and biological stabilities have beenevaluated in vitro in aqueous solutions and in rat blood, plasma, andlung and liver homogenates. In vitro anticholinergic activities werecharacterized through M₁-M₄ receptor binding affinities (pK_(i)), andthrough guinea pig ileum assay pA₂ values. In vivo pharmacologicalactivities were evaluated through mydriatic effects in rabbits andcardiac effects in rats. Pharmacokinetics after i.v. administration inrats has also been evaluated.

Material and Methods

Materials

Glycopyrrolate (glycopyrronium bromide) was kindly provided byBoehringer Ingelheim Chemicals, Inc. Carbamylcholine bromide(carbachol), atropine methylbromide (atropine MeBr), and scopolaminemethylbromide (scopolamine MeBr) were obtained from Sigma Chemicals Co.(St. Louis, Mo.); tropicamide (1%) was obtained from Bausch & LombPharmaceutical (Tampa, Fla.). N—[³H]-Methyl-scopolamine (NMS) wasobtained from Amersham Biosciences UK Limited (Buckinghamshire, UK).Cloned human muscarinic receptor subtypes M₁-M₄ were obtained fromApplied Cell Science Inc. (Rockville, Md.). Scintiverse BD was fromFisher Scientific Co. (Pittsburgh, Pa.). Animal studies were conductedin accordance with the Guide for the Care and Use of Laboratory Animalsadopted by the National Institute of Health. Institutional animal careand use committee (IACUC) approval was obtained prior to the initiationof this research and during its execution.

Synthesis

Racemic cyclopentylmandelic acid (1)

Cyclopentylmagnesium bromide ether solution (100 ml, 2M; 0.2 mol) wasadded drop-wise to benzoylformic acid (15 g, 0.1 mol) in 330 ml ofanhydrous ethyl ether at 0° C. The mixture was stirred at 0° C. for 30min and at room temperature for 24 h. The reaction mixture was treatedwith 1 N HCl, and the aqueous solution was extracted with ether. Thecombined ether solution was treated with K₂CO₃ solution. The potassiumcarbonate solution was acidified with HCl and extracted with ethertwice. The ether solution was dried with anhydrous sodium sulfate andevaporated to give a crude product. The crude product was washed withwater to get pure racemic cyclopentylmandelic acid 1 (8.0 g, 36.4%).Needle-like crystals, m.p.: 153-154° C. ¹H NMR (CDCl₃, 500 MHz):1.28-1.39, 1.42-1.50, 1.51-1.61, 1.63-1.72 [8H, m, (CH₂)₄], 2.93 [1H, p,CHC(OH)], 7.26-7.30, 7.33-7.36, 7.65-7.67 (5H, m, Ph) ppm.

Methyl cyclopentylmandelate (2)

To a mixture of racemic cyclopentylmandelic acid R/S(±)1 (4.47 g, 20mmol) and potassium carbonate (7.01 g, 50 mmol) in DMF (50 ml), methyliodide (8.64 g, 60 mmol) was added at room temperature. The mixture wasstirred at room temperature for 2 h, and then poured into water andextracted with hexanes three times. Evaporation of the dried hexanesextract gave a crude product. Flash chromatography of the crude producton silica gel with 1.5:1 hexanes:methylene chloride gave the pureproduct 2 (3.02 g, 64%). ¹H NMR (CDCl₃, 300 MHz): 1.32-1.37, 1.43-1.69[8H, m, (CH₂)₄], 2.90 [1H, p, CHC(OH)], 3.74 (1H, s, OH), 3.77 (3H, s,CH₃), 7.25-7.37, 7.63-7.65 (5H, m, Ph) ppm.

N-Methyl-3-pyrrolidinyl cyclopentylmandelate (4)

A solution of 2 (2.20 g, 9.4 mmol) and N-methyl-3-pyrrolidinol (3, 1.30g, 13 mmol) in 40 ml of n-heptane was heated until 20 ml of heptane hadbeen distilled. About 0.003 g of sodium was added, and the solution wasstirred and heated for 2 h as the distillation was continued. Moreheptane was added at such a rate as to keep the reaction volumeconstant. Additional sodium was added at the end of an hour. Thesolution was then cooled and extracted with 3N HCl. The acid extract wasmade alkaline with concentrated NaOH and extracted three times withether. Removal of the dried ether solution gave a crude oil. Flashchromatography of the crude product on silica gel with 8:1 EtOAc:EtOHgave pure product 4 (2.053 g, 72%). Analysis for C₁₈H₂₅NO₃. Calcd: C,71.26; H, 8.31; N, 4.62. Found: C, 71.55; H, 8.44; N, 4.68. ¹H NMR(CDCl₃, 500 MHz): 1.27-1.35, 1.40-1.47, 1.54-1.60, 1.75-1.90 [8H, m,(CH₂)₄], 2.12-2.30, 2.52-2.57, 2.64-2.81 (6H, m CH₂NCH₂CH₂), 2.33, 2.36(3H, 2s, NCH₃), 2.93 [(1H, p, CHC(OH)], 3.83 (1H, bs, OH), 5.23 (1H, m,CO₂CH), 7.23-7.36, 7.64-7.67 (5H, m, Ph) ppm.

3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, Compound (a)

To compound 4 (0.8235 g, 2.71 mmol) in 30 ml of dry acetonitrile, methylbromoacetate (1.08 g, 7.06 mmol) was added at room temperature. Themixture was stirred for 2 h. Evaporation of acetonitrile gave a crudeproduct. The crude product was dissolved in a small volume of methylenechloride and then poured into 100 ml of dry ethyl ether to precipitate.This procedure was repeated three times to obtain Compound (a) as pureproduct (0.9912 g, 80%). White powder, m.p.: 192-194° C. Analysis forC₂₁H₃₀BrNO₅. Calcd: C, 55.27; H, 6.63; N, 3.07. Found: C, 55.11; H,6.59; N, 3.03. ¹HNMR (CDCl₃, 500 MHz): 1.23-1.29, 1.31-1.37, 1.41-1.47,1.53-1.67 [8H, m, (CH₂)₄], 2.18-2.23, 2.73-2.80, 4.04-4.16, 4.21-4.25(6H, m, CH₂NCH₂CH₂), 2.85 [1H, p, CHC(OH)], 3.57 (3H, s, NCH₃), 3.80(3H, s, CO₂CH₃), 4.66, 4.85 (2H, 2dd, CH₂CO₂), 5.27 (1H, s, OH), 5.52(1H, m, CO₂CH), 7.25-7.28, 7.32-7.35, 7.57-7.59 (5H, m, Ph) ppm.

3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, Compound (b)

To compound 4 (0.369 g, 1.22 mmol) in 10 ml of dry acetonitrile, ethylbromoacetate (0.377 g, 2.25 mmol) was added at room temperature. Themixture was stirred for 2 h. Evaporation of acetonitrile gave a crudeproduct. The crude product was dissolved in a small volume of ethylenechloride and then poured into 50 ml of dry ethyl ether to precipitate.This procedure was repeated three times to obtain Compound (b) as pureproduct (0.45 g, 79%). White powder, m.p.: 192-194° C.

Analysis for C₂₂H₃₂BrNO₅. Calcd: C, 56.17; H, 6.86; N, 2.98. Found: C,56.14; H, 6.89; N, 2.94. ¹H NMR (CDCl₃, 500 MHz): 1.35 (3H, t, CH₃CH₂),1.26-1.33, 1.42-1.47, 1.55-1.67 [8H, m, (CH₂)₄], 2.14-2.21, 2.73-2.79,4.12-4.17, 4.22-4.29 (6H, m, CH₂NCH₂CH₂), 2.86 [1H, p, CHC(OH)], 3.62(3H, s, NCH₃), 4.25 (2H, q, CH₃CH₂), 4.67, 4.83 (2H, dd, CH₂CO₂), 4.91(1H, s, OH), 5.53 (1H, m, CO₂CH), 7.25-7.27, 7.32-7.34, 7.57-7.59 (5H,m, Ph) ppm.

Resolution of racemic cyclopentylmandelic acid (1)

(−)-Strychnine (6.10 g) in 50 ml of methanol (suspension) was added toracemic cyclopentylmandelic acid 1, (3.96 g) in methanol (20 ml) at roomtemperature. The reaction solution was let to stand for overnight. Thecrystals were removed by filtration and crystallized again with hotmethanol. The second crop of crystals was collected by filtration andtreated with sodium hydroxide solution. The basic solution was extractedwith methylene chloride twice (methylene chloride solution discarded),and then acidified with hydrochloric acid to recover the resolvedcyclopentylmandelic acid. To this resolved acid (20.6 mg in 0.1 ml ofethyl acetate), 13 μL of (+)-α-phenylethylamine was added. Theprecipitate which formed was washed with hexane three times and driedunder vacuum. The precipitate was identified by NMR as optically purecyclopentylmandelic acid, R(−)1, (1.49 g, 37.6%). M.p.: 121-122° C.[α]^(25°) _(D)=−22.5° (c=1 g/100 ml, CHCl₃). ¹H NMR (CDCl₃, 500 MHz):1.28-1.39, 1.42-1.50, 1.51-1.61, 1.64-1.73 [8H, m, (CH₂)₄], 2.93 [1H, p,CHC(OH)], 7.25-7.28, 7.32-7.35, 7.64-7.65 (5H, m, Ph) ppm.

Methyl(−)-cyclopentylmandelate, R(−)2

To a mixture of (−)-cyclopentylmandelic acid, R(−)1, (1.83 g, 8.3 mmol)and potassium carbonate (2.87 g, 21 mmol) in DMF (21 ml), methyl iodide(3.53 g, 25 mmol) was added at room temperature. The mixture was stirredat room temperature for 2 h, and then poured into water and extractedwith hexanes three times. Evaporation of the dried hexanes extract gavea crude product. Flash chromatography of the crude product on silica gelwith 1.5:1 hexanes:methylene chloride gave pure product R(−)2 (1.95 g,100%). Analysis for C₁₈H₁₈O₃. Calcd: C, 71.77; H, 7.74. Found: C, 71.88;H, 7.80. ¹H NMR (CDCl₃, 500 MHz): 1.32-1.36, 1.43-1.61 [8H, m, (CH₂)₄],2.90 [1H, p, CHC(OH)], 3.71 (1H, s, OH), 3.79 (3H, s, CH₃), 7.25-7.28,7.31-7.35, 7.63-7.65 (5H, m, Ph) ppm.

N-Methyl-3-pyrrolidinyl(−)-cyclopentylmandelate, 2R-4

A solution of R(−)2 (1.85 g, 7.9 mmol) and N-methyl-3-pyrrolidinol (3,1.05 g, 10.4 mmol) in 40 ml of n-heptane was heated until 20 ml ofheptane had distilled. Approximately 0.003 g of sodium was added, andthe solution was stirred and heated for 2 h as the distillation wascontinued. More heptane was added at such a rate as to keep the reactionvolume constant. Additional sodium was added at the end of an hour. Thesolution was then cooled and extracted with 3N HCl. The acid extract wasmade alkaline with concentrated NaOH and extracted three times withether. Removal of dried ether solution gave a crude oil. Flashchromatography of the crude product on silica gel with 8:1 EtOAc:EtOHgave 2R-4 as a mixture of two diastereoisomers in an NMR-estimated ratioof 1:1, (1.68 g, 70%). Analysis for C₁₈H₂₅NO₃.0.2H₂O. Calcd: C, 70.42;H, 8.34; N, 4.5. Found: C, 70.60; H, 8.26; N, 4.63. ¹H NMR (CDCl₃, 500MHz): 1.28-1.37, 1.40-1.47, 1.51-1.70, 1.73-1.80, 1.83-1.90 [8H, m,(CH₂)₄], 2.14-2.21, 2.27-2.35, 2.36-2.42, 2.52-2.55, 2.64-2.81 (6H, m,CH₂NCH₂CH₂), 2.33, 2.37 (3H, 2s, NCH₃), 2.93 [1H, p, CHC(OH)], 3.78 (1H,bs, OH), 5.22 (1H, m CO₂CH), 7.24-7.27, 7.31-7.35, 7.64-7.66 (5H, m, Ph)ppm.

(2R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, Compound (c)

To compound 2R-4 (0.15 g, 0.49 mmol) in 6 ml of dry acetonitrile, methylbromoacetate (0.194 g, 1.27 mmol) was added at room temperature. Themixture was stirred for 6 h. Evaporation of acetonitrile gave a crudeproduct. The crude product was dissolved in a small volume of methylenechloride and then poured into 50 ml of dry ethyl ether to precipitate.This procedure was repeated three times to obtain the product, Compound(c) (0.1879 g, 83%), as a mixture of four diastereoisomers in anNMR-estimated ratio of 1:1:2:2. White powder, m.p.: 153-155° C.[α]^(25°) _(D)=+0.50° (c=1 g/100 ml CHCl₃). Analysis forC₂₁H₃₀BrNO₅.0.2H₂O. Calcd: C, 54.86; H, 6.62; N, 3.05. Found: C, 54.75;H, 6.66; N, 3.01. ¹H NMR (CDCl₃, 500 MHz): 1.30-1.37, 1.41-1.50,1.55-1.73 [8H, m, (CH₂)₄], 1.93-2.00, 2.12-2.26, 2.75-2.95, 3.00-3.03,4.30-4.50, 4.57-4.61 [7H, m, CHC(OH) and CH₂NCH₂CH₂], 3.09, 3.30 (1H,2s, OH), 3.64, 3.66, 3.84, 3.95, 3.97 (3H, 5s, NCH₃), 3.74, 3.77, 3.79,3.81 (3H, 4s, CO₂CH₃), 4.78, 4.83; 4.90, 4.97; 5.30, 5.35; 5.37, 5.41(2H, 4 groups of 2dd, CH₂CO₂), 5.53 (1H, m, CO₂CH), 7.23-7.29,7.31-7.38, 7.56-7.60 (5H, m, Ph) ppm.

(2R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, Compound (d)

To compound 2R-4 (0.22 g, 0.73 mmol) in 10 ml of dry acetonitrile, ethylbromoacetate (0.21 ml, 0.316 g, 1.89 mmol) was added at roomtemperature. The mixture was stirred for 22 hours. Removal ofacetonitrile gave a crude product. The crude product was dissolved insmall volume of ethylene chloride and then poured into 50 ml of dryethyl ether to precipitate. This procedure repeated three times toobtain the product, Compound (d) (0.3085 g, 90%) as a mixture of fourdiastereoisomers in an NMR-estimated ratio of 1:1:2:2. White powder,m.p.: 143-145° C. [α]^(25°) _(D)=+5.60° (c=1 g/100 ml CHCl₃). Analysisfor C₂₂H₃₂BrNO₅.0.3H₂O. Calcd: C, 55.53; H, 6.91; N, 2.94. Found: C,55.46; H, 6.85; N, 2.97. ¹H NMR (CDCl₃, 500 MHz): 1.26, 1.28, 1.32, 1.35(3H, 4t, CH₃CH₂), 1.44-1.50, 1.53-1.63, 1.65-1.70 [8H, m, (CH₂)₄],1.93-2.00, 2.04-2.11, 2.18-2.25, 2.76-2.96, 3.01-3.04, 4.09-4.26 [7H, m,CHC(OH) and CH₂NCH₂CH₂], 3.06, 3.28 (1H, 2s, OH), 3.66, 3.69, 3.81,3.82, 3.94, 3.96 (3H, 6s, NCH₃), 4.61, 4.69; 4.76, 4.85; 5.17, 5.22;5.26, 5.30 (2H, 4 set of dd, CH₂CO₂), 4.26-4.52 (2H, m, CH₃CH₂), 5.53(1H, m, CO₂CH), 7.24-7.29, 7.31-7.38, 7.56-7.60 (5H, m, Ph) ppm.

Preparation of (±)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt [Compound (bb)] and (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt [Compound (cc)]

Both the racemic and isomeric acids were prepared by hydrolysis from thecorresponding methyl esters, (±) and (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, that have been synthesized and characterized (elementalanalysis, NMR) as described above. To Compounds (bb) and (cc) in aqueoussolutions, equimolar ratios of 0.1 N NaOH were added. The mixture wasstirred at room temperature for 3 h, and completion of reaction wasverified by HPLC. After volume adjustment by water, a 1% solution ofCompound (bb), or Compound (cc), pH about 6.5, was obtained. Theresulting solution was used as is or diluted with normal saline for theexperiments.

Analytical Methods

A high performance liquid chromatographic (HPLC) method was developedfor the quantitative analysis of Compound (cc). The system consisted ofa Spectra Physics (San Jose, Calif.) SP 8810 isocratic pump, a SP 8450UV/V is detector (wavelength set to 230 nm), a SP 4290 integrator, and aSupelco Discovery C16 column. The mobile phase consisted ofacetonitrile, water, and acetic acid at a ratio of 30:70:0.1. At a flowrate of 1 mL/min and an injection volume of 10 μL, the retention timewas 7.10 min, and the detection limit was 1 μg/mL.

Stability Studies

Stability in Aqueous Solutions

A 0.1% water solution of Compound (cc) (pH 6.5) was kept at roomtemperature or 37° C. At various time points, samples were withdrawn andanalyzed by HPLC.

Stability in Biological Media

Freshly collected rat blood, plasma, and 30% liver and lung homogenateswere used. Aliquots of 1% of Compound (cc) in water solution were addedto the biological mediums at 37° C., to yield final concentrations of0.1%. At appropriate time intervals, samples (0.1 mL) were withdrawn andmixed with 0.2 mL of 5% dimethylsulfoxide in acetonitrile solution. Themixtures were centrifuged, and the supernatants were further diluted twotimes by water and analyzed by HPLC. The extraction rate was compared toa calibration standard and determined to be 100±3% (n=4).

In Vitro Pharmacodynamic Evaluations

Receptor Binding Affinity

Receptor binding studies on Compound (bb), Compound (cc),glycopyrrolate, and N-methylscopolamine were performed withN—[³H]-methyl-scopolamine (NMS) in assay buffer (phosphate-bufferedsaline, PBS, without Ca⁺⁺ or Mg⁺⁺, pH 7.4), following the protocol fromApplied Cell Science Inc. (Rockville, Md.). A 10 mM NaF solution wasadded to the buffer as an esterase inhibitor. The assay mixture (0.2 mL)contained 20 μL diluted receptor membranes (receptor proteins: M₁, 38μg/mL; M₂, 55 μg/mL; M₃, 27 μg/mL; M₄, 84 μg/mL). The finalconcentration of NMS for the binding studies was 0.5 nM. Specificbinding was defined as the difference in [³H]NMS binding in the absenceand presence of 5 μM atropine for M₁ and M₂ or 1 μM atropine for M₃ andM₄. Incubation was carried out at room temperature for 120 min. Theassay was terminated by filtration through a Whatman GF/C filter(presoaked overnight with 0.5% polyethyleneimine). The filter was thenwashed six times with 1 mL ice cold buffer (50 mM Tris-HCl, pH 7.8, 0.9%NaCl), transferred to vials, and 5 mL of Scintiverse was added.Detection was performed on a Packard 31800 liquid scintillation analyzer(Packard Instrument Inc., Downer Grove, Ill.). Data obtained from thebinding experiments were fitted to the %[³H]NMSbound=100−[100x^(n)/k/(1+x^(n)/k)] equation, to obtain the Hillcoefficient n, and then to %[³H] NMSbound=100−[100x^(n)/IC₅₀/(1+x^(n)/IC₅₀)], to obtain the IC₅₀ values (xbeing the concentration of the tested compound). Based on the method ofCheng and Prusoff [Biochem. Pharmacol. 22: 3099-3108 (1973)], K_(i) wasderived from the equation K_(i)=IC₅₀/(1+L/K_(d)), where L is theconcentration of the radioligand. IC₅₀ represents the concentration ofthe drug causing 50% inhibition of specific radioligand binding, andK_(d) represents the dissociation constant of the radioligand receptorcomplex. Data were analyzed by a non-linear least-square curve-fittingprocedure using Scientist software (MicroMath Inc., Salt Lake City,Utah).

Determination of pA₂ Values

Male guinea pigs obtained from Harlan Sprague Dawley Inc. (Indianapolis,Ind.) and weighing about 400 g were used after overnight fasting.Animals were sacrificed by decapitation, and the ileum (the region of 5cm upward of the cecum) was isolated and removed. The ileum was cut into2.5 cm pieces and suspended in an organ bath containing 30 mL of mixtureof Tyrode's solution and 0.1 mM hexamethonium bromide. The organ bathwas constantly aerated with oxygen and kept at 37° C. One end of theileum strip was attached to a fixed support at the bottom of the organbath, and the other end to an isometric force transducer (Model TRN001,Kent Scientific Corp., Conn.) operated at 2-10 g range. The ileum stripwas kept at a 2 g tension, and carbachol was used as antagonist. Theileum contracted cumulatively upon the addition of consecutive doses ofcarbachol (10-20 μL of 2×10⁻⁴-2×10⁻³ M in water solution). Contractionswere recorded on a physiograph (Kipp & Zonen Flarbed Recorder, Holland).After the maximum response was achieved, the ileum was washed threetimes, and a fresh Tyrode's solution containing appropriateconcentration of the antagonist [Compound cc), Compound (bb), Compound(c), glycopyrrolate, or scopolamine] was replaced. An equilibration timeof 10 min was allowed for the antagonists before the addition ofcarbachol. In each experiment, 5 to 6 different concentrations wereused, and a Schild plot was used to obtain the pA₂ values. Four to sixtrials were performed for each antagonist.

In Vivo Pharmacodynamic Evaluations

Mydriatic Studies

Topical administration. The mydriatic effects of Compound (cc) have beencompared to those of glycopyrrolate, tropicamide, and its parent softdrugs, Compounds (c) and (d), in rabbit eyes. Four healthy, maleNew-Zealand white rabbits weighting about 3.5 kg were used. Toinvestigate the dose-mydriatic-response relationships, 100 μL of variousconcentrations of the compounds (0-1%) were administered in the eyes todetermine the pharmacodynamically equivalent doses, the lowest dosesthat induce the maximum pupil dilations. Drug solutions were applied toone eye; only water was applied to the other eye that served as control.Experiments were carried out in a light- and temperature-controlledroom. At appropriate time intervals, the pupil diameters of both eyeswere recorded. Difference in pupil diameters between each time-point andzero time-point were calculated for both treated and control eyes andreported as mydriatic responses [(treated−control)/control in %].Control eye dilations were monitored to determine whether systemicabsorption had occurred or not. The area under the mydriaticresponse-time curve (AUC^(eff)) was calculated by the trapezoidal rule,and it was used to compare the activity and duration of action of thetested compounds.

Intravenous Administration.

New-Zealand white rabbits (4 kg) were injected i.v. with Compound (cc)or glycopyrrolate at a dose of 2.5 tμmol/kg (about 1 mg/kg), and themydriatic response was recorded for both eyes at various time points.

Cardiac Studies

Effect on Resting Heart Rate

Male Sprague-Dawley rats, weighing 300±30 g, were anesthetized with 50mg/kg (i.p.) of sodium pentobarbital. Needle electrodes were inserteds.c. into the limbs of the anesthetized rats and were joined to a GOULD2000 recorder (GOULD Inc., Cleveland, Ohio). Standard leads I, II, andIII were recorded at a paper speed of 25 mm/sec. After a 15 min periodof stabilization, baseline electrocardiography (ECG) was taken, and drugwas administrated. Compound (cc) in normal saline (5 μmol/kg, about 2mg/kg) or vehicle only, was administered in the jugular vein (1 mL/kg).Heart rate was recorded at designated time-points for 2.5 h.

Effect on Carbachol-Induced Bradycardia

Rats were prepared as previously described. Recording was taken before,during, and after the administration of any of the compounds, until allbasic ECG parameters returned to the baseline. ECG recordings wereevaluated for the following parameters: PP cycle length (msec), RR cyclelength (msec), heart rate (1/min) by the equation of 60000/RR cyclelength, and presence of Mobitz II type atrio-ventricular (A-V) block(2:1, 3:1, etc.). To evaluate the effects of carbachol, the negativechronotropic and dromotropic effects were analyzed. These effects ofcarbachol were manifested on the surface ECG as sinus bradycardia(lengthening of the PP cycle) and as a development of Mobitz II type A-Vblock. After analyzing the ECG recordings, the percent changes of heartrate, as compared to that of the baseline, were plotted against time,and the effects of drugs on the percent changes of the heart rate werecharacterized. Compound (cc) (0 to 5 μmol/kg) and glycopyrrolate (0.5μmol/kg) were dissolved in 0.9% NaCl and injected into the jugular veins(1 mL/kg) at time 0, while carbachol (80 μg/mL, 0.06-0.1 mL volumeaccording to the initial individual ECG response of each rat) wasinjected at various time-points after drug administration. Student'st-test was used for statistical evaluations.

Pharmacokinetic Studies

Pharmacokinetics after Intravenous Administration

Male Sprague-Dawley rats (body weight about 400 g) were anesthetizedwith 30 mg/kg of sodium pentobarbital (i.p.). A 1% solution of Compound(cc) was injected in the jugular vein. Due to the low sensitivity ofHPLC detection (1 μg/mL), a dose of 30 mg/kg was used. Blood samples,0.12 mL, were collected through heparinized syringe from thecontralateral jugular vein at appropriate time intervals, and plasma(0.05 mL) was separated. The plasma samples were mixed with 0.1 mL ofacetonitrile containing 5% dimethyl sulfoxide and centrifuged. Thesupernatants were further mixed with one volume of water, centrifuged,and analyzed by HPLC. The concentrations of Compound (cc) have beendetermined using a calibration curve obtained by addition of knownamounts of the compound to plasma and prepared following the samemethodology for HPLC analysis (r=0.995). Noncompartmental andcompartmental pharmacokinetic analysis was performed using WinNonlin(Pharsight Corp., Mountain View, Calif.). In noncompartmental analysis,the area under the curve (AUC) of the plasma concentration versus timewas calculated using the trapezoidal rule. The area from the lastconcentration measured (C_(t)) to infinity was calculated as C_(t)/β,where β is the terminal disposition rate constant. Total body clearance(CL_(tot)) was calculated as Dose/AUC. Mean residence time (MRT) wascalculated as AUMC/AUC, where AUMC, the area under the first momentcurve, was calculated using the trapezoidal rule from the graph ofplasma concentration×time vs. time. Extrapolation of AUMC from the lasttime point t to infinity was calculated as C_(t)/β+C_(t)/β². The volumeof distribution at the steady state (Vd_(ss)) was determined as theproduct of CL_(tot) and MRT. For compartmental analysis, data werefitted with a two-compartment model, C=A_(e) ^(−αt)+Be^(−βt), where C isthe drug concentration in plasma, A and B are the exponentialmultipliers, α and β are the hybrid rate constants for the distributionand elimination phase, respectively. AUC was calculated as A/α+B/β, andthe half-life of the terminal phase (t_(1/2)) was obtained as 1n2/β. Thevolume of distribution of the central compartment, Vd_(c), wascalculated as Dose/A+B. The elimination rate constant, K_(el), wascalculated as CL_(tot)/Vd_(c). Unweighted data were used in allanalyses.

Excretion after Intravenous Administration

Male Sprague-Dawley rats (350±10 g) were anesthetized by injection ofsodium pentobarbital (30 mg/kg, i.p.). The urinary tract was closed toprevent urination, and urine samples were collected directly from theurinary bladder through a 26 gauged needle. The peritoneal cavity wasexposed, and the bile duct was cannulated using a polyethylene tube (PE10). Compound (cc) (10 mg as 1 mL 1% solution) was administeredintravenously jugular vein). At various time intervals afteradministration, total bile juice and urine were collected and weighed incentrifuge tubes. Samples were taken at 0 min (control) and then every15 min until 2 h after i.v. administration. A 5% mannitol in normalsaline solution was injected (0.5 mL) in the jugular vein every 30 minto increase the volume of the urine for sample collections (about 0.2 mLper 15 min). The collected bile and urine samples were prepared, dilutedproperly, and analyzed by HPLC as described in the pharmacokineticstudies. Noncompartmental analysis for the urine data after bolus i.v.was performed using WinNonlin. Maximum observed excretion rate (C_(max))was recorded, and the first order elimination rate constant (k) wasestimated via linear regression using the logarithmic plot. The halflife (t_(1/2)) was calculated as 1n2/k, and the amount of cumulativeelimination, A_(e), was calculated by summation of theconcentration-volume products of each sample. Using the remainingamounts vs. time, a sigma (−) plot was also developed, and k and t_(1/2)were calculated subsequently for comparison.

Results and Discussion

Preparation of Zwitterions

Both Compound (cc) and its racemic equivalent Compound (bb) arehydrolytic products of soft anticholinergic compounds, (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, Compound (c), or the corresponding (±) compound, Compound (a).They were prepared by simple hydrolysis in basic aqueous solution of thecorresponding ester.

Solubility and Stability

Both zwitterionic Compound (cc) and Compound (bb) are very soluble inaqueous solutions (pH 6.5) and biological media, and they are also verystable. A 0.1% water solution kept at room temperature showed nodecomposition after one year. In freshly collected rat blood, plasma,and 30% liver and lung homogenates, there was no change in the HPLC peakarea after two hours at 37° C. Thus, Compound (cc) (0.1%) is also verystable toward metabolic transformations.

Pharmacodynamic Evaluations

Receptor Binding Affinity

The receptor binding affinities, pK_(i), of Compound (cc) and Compound(bb) are presented in Table 1 together with that of the parent softdrugs of Compound (cc), that is, the methyl ester Compound (cc) and theethyl ester Compound (d), and those of glycopyrrolate andN-methylscopolamine for comparison. The receptor binding affinity of thezwitterionic metabolite is considerably less than that of glycopyrrolateor N-methylscopolamine, and is about an order of magnitude less thanthat of their parent methyl ester soft drugs; i.e., Compound (cc) vs.Compound (c) (all differences statistically significant at the p<0.05level using either t-tests or nonparametric Mann-Whitney U tests). Thisis in good agreement with the hypothesis that presence of the acidicmoiety formed by hydrolysis of the parent soft drug ester inactivatesthe drug, but because in these zwitterionic structures, the electronicdistribution somewhat resembles those of the neutral (and active)anticholinergics, and thus significant activity is still retained.Contrary to its parents Compound (c) or Compound (d) that show no M₃/M₂subtype selectivity, Compound (cc) shows a significantly better (p<0.01,t-test assuming equal variances), almost five-fold subtype selectivity(Table 1 below), further increasing its safety profile. Furthermore,even on these structures, the 2R isomer shows increased affinityconfirming the stereospecificity of muscarinic receptors. Hillcoefficients (n) were not very different from unity indicating that, ingeneral, drug-receptor interactions obeyed the law of action and bindingtook place at only one site. The receptor binding affinity ofN-methylscopolamine determined here was in good agreement withpreviously published results.

TABLE 1 Receptor binding affinities, M_(3/M) ₂ selectivities, and pA₂values. Subtypes of cloned muscarinic receptors^(a) Selectivity Cpd. M₁M₂ M₃ M₄ M₃/M₂ pA₂ ^(b) Cpd (cc) 8.11 ± 0.16 7.48 ± 0.12 8.12 ± 0.108.23 ± 0.12 4.4 ± 0.3 7.20 ± 0.19 (0.83 ± 0.11) (1.10 ± 0.11) (0.83 ±0.01) (0.83 ± 0.01) Cpd (bb) 6.19 ± 0.06 5.48 ± 0.13 5.84 ± 0.07 6.44 ±0.06 2.3 ± 0.7 6.42 ± 0.30 (1.11 ± 0.06) (1.02 ± 0.20) (1.01 ± 0.07)(0.84 ± 0.06) Cpd (c) 8.89 ± 0.04 8.87 ± 0.05 9.00 ± 0.06 9.52 ± 0.011.4 ± 0.1 8.31 ± 0.05 (0.83 ± 0.11) (1.10 ± 0.11) (0.83 ± 0.01) (0.83 ±0.01) Cpd (d) 8.67 ± 0.16 8.84 ± 0.34 8.74 ± 0.02 8.85 ± 0.13 0.9 ± 0.68.55 ± 0.16 (0.86 ± 0.08) (0.92 ± 0.01) (1.09 ± 0.15) (0.89 ± 0.02)glycopyrrolate 9.76 ± 0.05 9.19 ± 0.18 8.73 ± 0.05 9.90 ± 0.08 0.4 ± 0.18.57 ± 0.12 (1.37 ± 0.20) (0.99 ± 0.11) (1.14 ± 0.25) (1.02 ± 0.01)scopolamine 9.69 ± 0.01 9.18 ± 0.21 9.29 ± 0.12 9.92 ± 0.21 1.3 ± 0.49.16 ± 0.19 methyl (0.92 ± 0.10) (1.02 ± 0.02) (1.07 ± 0.01) (0.90 ±0.04) bromide ^(a)Receptor binding pK_(i) data represent mean ± S.D. of3 experiments. The numbers in parentheses denote Hill slopes. ^(b)pA₂values were determined on 4-6 ileum strips obtained from differentanimals. Data represent mean ± S.D.Guinea Pig Ileum Assay, pA₂ Value

The pA₂ values determined from guinea pig ileum contraction assays,which represent the negative logarithm of the molar concentration of theantagonist that produces a two-fold shift to the right in an agonist'sconcentration-response curve, are a classical functional study ofanticholinergic affinity (at M₃ muscarinic receptors). Values obtainedfor the present compounds from ileum longitudinal contractions by usingcarbachol as agonists with the method of van Rossum (50) are presentedin Table 1 above. Compared to the parent esters Compound (c) and (d),glycopyrrolate, or N-methylscopolamine, pA₂ values indicate evensomewhat less activity for the zwitterionic Compound (cc) than thereceptor binding assays, and this assay also confirmed the higheractivity of the 2R isomers. The pA₂ values of the Compound (cc) and (bb)metabolites are 1.1±0.3 and 1.3±0.3 less than those of the correspondingethyl and methyl parent ester soft drugs, respectively indicating againthat they are a good order of magnitude less active. Comparison of thisto the average of close to two orders of magnitude decrease in activityseen previously in the same pA₂ assay for three other acidic metabolitesversus their corresponding ethyl ester parents, 1.8±0.5 (31-33),confirms the hypothesis that these spatially-close zwitterions arelikely to retain more activity than the previous metabolite structures,but are still inactivated to a good extent.

Mydriatic Studies

These studies were performed to evaluate the in vivo potency of thesemetabolites following local or systemic administration.

Topical Administration.

The potency and duration of action of Compound (cc) was compared tothose of its parent ester soft drugs Compounds (c) and (d),glycopyrrolate, and tropicamide (the most frequently used short actingmydriatic agent). Following topical administration of 100 μL drugsolution to one eye in rabbits, the pupil size was measured, and themaximum mydriatic effect (% change in pupil size) and area under themydriatic response-time curves (AUC^(eff) _(0-168h)) were determined.See Table 2.

TABLE 2 Maximum response (R_(max), maximum % change in pupil size) andarea under the response-time curve (AUC^(eff)) after topicaladministration (0.1 mL).^(a) Compound Conc. (%) R_(max) (%) AUC^(eff)_(0-168 h) Cpd (cc) 0 0.00 ± 0.00 0 ± 0 0.01 31.00 ± 7.14  73 ± 24 0.0238.79 ± 7.45  103 ± 22  0.05 51.38 ± 8.81  175 ± 46  0.1 50.34 ± 7.92 182 ± 40  0.2 55.65 ± 9.24  240 ± 38  0.5 56.79 ± 10.71 590 ± 205 153.65 ± 10.84 612 ± 115 Cpd (bb) 0.01 1.85 ± 2.14 0.7 ± 0.9 1 45.37 ±8.19  119 ± 34  Cpd (c) 0.5 52.92 ± 13.41 752 ± 342 1 57.08 ± 11.66 875± 197 Cpd (d) 0.5 53.96 ± 13.27 1170 ± 308  1 56.04 ± 11.69 1532 ± 526 glycopyrrolate 0.02 35.97 ± 9.84  1879 ± 664  0.05 48.73 ± 12.66 2476 ±847  0.1 52.95 ± 10.93 3732 ± 866  0.2 53.24 ± 14.49 4923 ± 2175tropicamide 0.02 30.27 ± 9.74  99 ± 40 0.05 35.00 ± 9.18  167 ± 116 0.242.72 ± 9.60  435 ± 150 0.5 44.64 ± 11.17 622 ± 171 ^(a)Data representmean ± SD of four trials.

Accordingly, it was found that Compound (cc) produces local mydriaticactivity (R_(max)), but only with short duration of action (AUC^(eff)_(0-168h)). See FIG. 1. Recovery times, time-periods needed for the sizeof pupil in the treated eye to recover within less than 1 mm of thecontrol, were approximately 102, 24, and 6 h after administration of0.2% of glycopyrrolate, 1.0% Compound (d), and Compound (cc),respectively. Compound (cc) was less potent and shorter acting than itsparent esters, Compound (a) and Compound (d). In agreement with previousresults, its racemic form Compound (bb) showed even lower potency.Furthermore, Compound (cc) did not cause any observable irritationreactions, such as eye-closing, lacrimation, or mucous discharge; andunlike conventional anticholinergics, it did not cause pupil dilation inthe contralateral, untreated eye, indicating not only low topical andsystemic side effects, but also rapid elimination from the systemiccirculation.

Intravenous Administration.

To evaluate the likelihood of causing side effects after systemicadministration, the mydriatic response following an intravenous (i.v.)dose of 2.5 μmol/kg (about 1 mg/kg through ear vein) also wasinvestigated in rabbits. As shown in FIG. 2, Compound (cc) produced somemydriasis after i.v. administration, but its magnitude and duration ofaction were much less than those produced by glycopyrrolate.

Cardiac Studies

Effect on Resting Heart Rate.

After i.v. administration of normal saline (vehicle control) or Compound(cc) in pentobarbital-anesthetized rats, the heart rate was recordedevery 10 min up to 2.5 h. Results indicate that at a dose of 5 μmol/kg(about 2 mg/kg), Compound (cc) did not affect the resting heart rate inany of the four animals, further confirming its slight subtypeselectivity. Results are depicted in FIG. 3.

Effect on Carbachol-Induced Bradycardia.

The magnitude of cardiac effects of Compound (cc) and glycopyrrolatewere assessed by measuring the extent and duration of their bradycardiaprotecting activities. Intravenous injection of carbachol at a dose of16-26 μg/kg to rats produces a temporary sinus bradycardia and Mobitz IIA-V block in a safe and reproducible manner. This can be fully preventedby prior administration of an anticholinergic agent, and the effects ofvarious anticholinergics differ greatly in their extent and duration ofaction. In this study, various doses (0.25-5 μmol/kg) of Compound (cc)have been investigated and compared to glycopyrrolate (0.5 μmol/kg). Asshown in FIG. 4, carbachol injection (e.g., at −5 min) induced atemporary Mobitz II A-V block with more than 60% inhibition of thenormal heart rate (control). After i.v. injection at 0 min of Compound(cc) or glycopyrrolate at various doses, carbachol at the same doseinduced various degrees of inhibition. The zwitterionic Compound (cc)showed bradycardia-protecting activity immediately after i.v.administration, but this diminished rapidly. At a dose of 0.5 μmol/kg,two out of three rats showed full prevention of the carbachol inducedbradycardia at 1 min, and at a dose of 1.25 μmol/kg, all three ratsshowed full prevention. These protecting effects disappeared completelyin less than 30 and 60 min, respectively. Even at higher doses, such as2.5 and 5 μmol/kg, the effect of Compound (cc) disappeared in less than100 min indicating that fast elimination from the systemic circulationrapidly reduces the potential to induce heart-related side effects. Forcomparison, glycopyrrolate showed full protection for more than 2.5 hand partial effect for another 1.5 h even at a ten-times smaller dose(0.5 μmol/kg).

Pharmacokinetic Studies

Pharmacokinetics after Intravenous Administration

An in vivo pharmacokinetic evaluation of Compound (cc) was performed inrats. After a single i.v. bolus injection, plasma concentrations atpredetermined time points were quantified by HPLC. The observed plasmaconcentration time-profile of Compound (cc) (FIG. 5) could be bestdescribed by a two-compartmental body model corresponding to abi-exponential equation, C=Ae^(αt)+Be^(−βt). Pharmacokinetic parametersobtained from non-compartmental (NCA) and compartmental analyses areshown in Table 3 below. The elimination half-life t_(1/2) was about 10.7min with a volume of distribution Vd_(ss) of 193 mL/kg and a total bodyclearance CL_(tot) of about 20 mL/min/kg (NCA). For the compartmentalanalysis, the correlation coefficient, r, was >0.999 in all individuals.Parameters were in general agreement with the NCA results. These clearlydemonstrate that even a high dose (30 mg/kg) of Compound (cc) is rapidlycleared and well-tolerated in animals, an important requirement for softdrug metabolites.

TABLE 3 Pharmacokinetics of Compound (cc) after i.v. bolusadministration in rats. Parameter Mean SD Dose, mg/kg 30Noncompartmental analysis results C_(max), μg/mL 330.74 31.96 k_(e),min⁻¹ 0.066 0.01 t_(1/2), min 10.7 1.40 AUC_(∞), (μg · min/mL) 1676 637CL_(tot), (mL/min)/kg 19.59 6.05 AUMC_(∞), (μg · min²/mL) 18281 10850MRT_(∞), min 10.33 2.12 Vd_(ss), mL/kg 192.99 30.94 Compartmentalanalysis results A, μg/mL 292.46 25.37 B, μg/mL 111.41 39.71 α, min⁻¹0.851 0.145 β, min⁻¹ 0.094 0.012 t_(1/2α), min 0.832 0.14 t_(1/2β), min7.44 0.96 Vd_(c), mL/kg 75.20 9.86 Vd_(ss), mL/kg 167.91 22.76 r 0.99940.0005Excretion after Intravenous Administration

Total bile juice and urine were collected every 15 min for up to 2 h andanalyzed by HPLC after i.v. administration of a dose of 10 mg ofCompound (cc) (1 mL of 1% solution). No detectable levels of Compound(cc) were excreted in the bile, but relatively large amounts of Compound(cc) were excreted in the urine. Table 4 below presents the results ofnon-compartmental analysis of the urine data (log rate plot and sigmaminus plot). At one hour after injection, the cumulative excretionamount was about 50% of the administered dose. The first orderelimination rate constants k_(e) were estimated, and the eliminationhalf lives t_(1/2) were derived from the slopes of these two plots (14.0and 13.1 min, respectively). These results, again, indicate a rapidelimination of Compound (cc) from the systemic circulation mainlythrough urinary excretion.

TABLE 4 Excretion of Compound (cc) in urine after i.v. bolusadministration in rats. Parameter Mean SD Dose, mg 10 Log rate plotC_(max), mg/mL 0.193 0.046 A_(e), mg (cumin. excr.) 5.08 0.46 k_(e),min⁻¹ 0.050 0.0062 t_(1/2), min 14.02 1.73 AUC_(0-last), (mg/mL) × min4.36 0.34 AUC_(∞), (mg/mL) × min 4.46 0.31 r 0.974 0.021 Sigma minusplot k_(e), min⁻¹ 0.0539 0.0089 t_(1/2), min 13.13 2.22 r 0.995 0.003Conclusion

The present PK/PD studies demonstrated that the zwitterionic metaboliteCompound (cc) retains significant but reduced activity of its parentquaternary ammonium ester soft drugs, and is very rapidly eliminatedfrom the systemic circulation mainly through urinary excretion of itsunchanged form. Furthermore, as Compound (cc) also showed moderate M₃/M₂muscarinic receptor subtype selectivity, the likelihood of cardiac sideeffects is further reduced for this metabolite.

Further Studies

Purpose. Because stereospecificity is known to be important atmuscarinic receptors, isomers of both N-substituted softanticholinergics based on glycopyrrolate, (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(alkoxycarbonylmethyl)-1-methylpyrrolidiniumbromide methyl and ethyl esters, Compounds (c) and (d), and theirzwitterionic metabolite, (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-carboxymethylpyrrolidiniuminner salt [Compound (cc)], were synthesized and their pharmacologicalactivities were evaluated in vitro and in vivo.

Methods. The isomers of Compounds (c) and (d) were synthesized with bothoptically pure methyl-cyclopentylmandelate and3-hydroxy-N-methylpyrrolidine. Trans-esterification followed byquaternization with alkyl bromoacetate gave four isomers of the methylor ethyl ester with the nitrogen chiral center unresolved. Thehydrolysis of these four isomers followed by HPLC separation resulted ineight fully resolved isomers of the corresponding acid. Thepharmacological activities were assessed using the in vitroreceptor-binding assay, guinea pig ileum pA₂-assay, and in vivo rabbitmydriatic effect. The results were compared with that of conventionalanticholinergic agents such as glycopyrrolate, N-meythylscopolamine, andtropicamide as well as that of previously prepared racemates and 2Risomers.

Results. The receptor binding at cloned human muscarinic receptors(M₁-M₄ subtypes), pK_(i) values, of these newly synthesized methyl andethyl ester isomers were in the 6.0-9.5 range, and zwitterion isomers in5.0-8.6 range. In both cases, 2R isomers were found significantly moreactive than 2S isomers (27-447 times for methyl ester isomers, and 6 to4467 times for zwitterion isomers). Among four isomers of the methylester Compound (c) (with chiral center 1′ unresolved), the 3′R isomerswere more active than the corresponding 3′S isomers (1.5-12.9 times).However, in the case of zwitterion isomers, the 3′S isomers were notalways more active than the corresponding 3′R isomers, indicating thatactivity determined based on chiral center 3′ is significantly affectedby the configuration of other two chiral centers, 2 and 1′. In thecompletely resolved 8 zwitterion isomers (all the three chiral centersresolved), it was found that 1′S isomers were more active than thecorresponding 1′R isomers in all cases (1.8-22.4 times). The resultsalso indicate that some isomers showed good M₃/M₂ muscarinic-receptorsubtype-selectivities (about 3-5 times), and 2R and 3′S were thedetermining configurations for this property. Guinea pig ileum assaysand rabbit mydriasis test on zwitterion isomers also confirmed thestereospecificity. In rabbit eyes, some 2R-zwitterion isomers showedmydriatic potencies similar to glycopyrrolate and exceeded tropicamide,but their mydriatic effects lasted considerably less time, and they didnot induce dilation of the pupil in the contralateral, water-treatedeyes. These results indicate that, in agreement with their soft nature,they are locally active, but safe and have a low potential to causesystemic side effects. The pharmacological potency of eight zwitterionisomers was concluded to be (2R1′S3′S, 2R1′S3′R and2R1′R3′S)>2R1′R3′R>2S1S3′R>(2S1′S3′S and 2S1 ′R3′R)>2S1′R3′S.

Conclusions. The stereospecificity and M₃/M₂ muscarinic-receptorsubtype-selectivity of soft anti-cholinergics, Compounds (c) and (d) andtheir zwitterionic metabolite Compound (cc), have been demonstrated.Adding to the previous results, safe use of these soft drugs has beenconfirmed.

INTRODUCTION

Stereospecificity of anticholinergics is important at muscarinicreceptors. Compounds (c) and (d), (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(alkoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, and their common zwitterionic metabolite, (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-carboxymethylpyrrolidiniuminner salt, have shown promising activity and safety in animal studies.These compounds indeed exhibited stereospecificity toward muscarinicreceptors, and the anticholinergic activity has been improved with the2R configuration. In addition, the zwitterionic metabolite also showed amoderate M₃/M₂ muscarinic-receptor subtype-selectivity that indicates areduced systemic cardiac side effect. However, the molecules of thistype of soft analogs possess three chiral centers, so that each racemiccompound may contains up to eight different isomers, that is 2R1′R3′R,2R1′R3′S, 2R/S3′R, 2R1′S3′S, 2S1′R3′R, 2S1′R3′S, 2S1′S3′R, and 2S1′S3′Sas displayed below:

Thus, the above-described investigations in the soft glycopyrrolateisomers based on one resolved chiral center (2R or 2S) only expressedthat 2R enantiomers (a mixture of four diastereoisomers 2R1′3′R,2R1′R3′S, 2R1′S3′R, 2R1′S3′S) were more active than 2S enantiomers (amixture of 2S1′R3′R, 2S1′R3′S, 2S1′S3′R, 2S1′S3′S). In this section,further investigations in the stereospecificity of these softglycopyrrolates are reported using five partially-resolved softanticholinergic ester isomers and eight fully resolved zwitterionmetabolite, isomers (as described for 2R and 2S enantiomers). Thecompounds were systematically synthesized and isomers were separated.The relative pharmacological activities and M₃/M₂ muscarinic-receptorsubtype-selectivities were investigated by in vitro receptor-bindingassay, in vitro guinea pig ileum pA₂-assay, and in vivo mydriatic effectin rabbits.

Materials and Methods

Materials

Glycopyrrolate (glycopyrronium bromide) was kindly provided byBoehringer Ingelheim Chemicals, Inc. Carbamylcholine bromide(carbachol), atropine methylbromide (atropine MeBr), and scopolaminemethylbromide (scopolamine MeBr) were obtained from Sigma Chemicals Co.(St. Louis, Mo.), and tropicamide (1%) was obtained from Bausch & LombPharmaceutical (Tampa, Fla.). N—[³H]-Methyl-scopolamine (NMS) wasobtained from Amersham Biosciences UK Limited (Buckinghamshire, UK).Cloned human muscarinic receptor subtypes M₁-M₄ were obtained fromApplied Cell Science Inc. (Rockville, Md.). Scintiverse BD was fromFisher Scientific Co. (Pittsburgh, Pa.). (R)-3-hydroxy pyrrolidinehydrochloride and (S)-3-hydroxy pyrrolidine hydrochloride were fromAstatech Inc. (Monmouth Junction, N.J.). N—[³H]-Methyl-scopolamine (NMS)was from Amersham Biosciences UK Limited (Buckinghamshire, UK). Clonedhuman muscarinic receptor subtypes M₁-M₄ were from Applied Cell ScienceInc. (Rockville, Md.). Scintiverse BD was from Fisher Scientific Co.(Pittsburgh, Pa.). Other chemicals used for synthesis were reagent orHPLC grade, and were obtained from Aldrich (Milwaukee, Wis.) and FisherScientific Co. Melting points were taken on Fisher-Johns meltingapparatus. NMR spectra were recorded on Bruker Advance 300, 400 and 500MHz NMR spectrometers, and are reported in ppm relative to TMS. NOESYwas performed using 2D NMR spectrometer, Mercury-300BB. Animal studieswere performed in accordance with the Guide for the Care and Use ofLaboratory Animals adopted by the National Institute of Health, USA.Institutional animal care and use committee (IACUC) approval wasobtained prior to the initiation of this research and during itsexecution.

Synthesis of 2R-isomers

Racemic cyclopentylmandelic acid, 1

Cyclopentylmagnesium bromide ether solution (100 ml, 2M; 0.2 mol) wasadded drop-wise to benzoylformic acid (15 g, 0.1 mol) in 330 ml ofanhydrous ethyl ether at 0° C. The mixture was stirred at 0° C. for 30min and at room temperature for 24 h. The reaction mixture was treatedwith 1 N HCl, and the aqueous solution was extracted with ether. Thecombined ether solution was treated with K₂CO₃ solution. The potassiumcarbonate solution was acidified with HCl and extracted with ethertwice. The ether solution was dried with anhydrous sodium sulfate andevaporated to give a crude product. The crude product was washed withwater to get pure racemic cyclopentylmandelic acid 1 (8.0 g, 36.4%).Needle-like crystal, m.p.: 153-154° C. ¹H NMR (CDCl₃, 300 MHz):1.28-1.39, 1.42-1.50, 1.51-1.61, 1.63-1.72 [8H, m, (CH₂)₄], 2.93 [1H, p,CHC(OH)], 7.26-7.30, 7.33-7.36, 7.65-7.67 (5H, m, Ph) ppm.

Resolution of racemic cyclopentylmandelic acid, R(−)1

(−)-Strychnine (11.4 g) in 100 ml of methanol (suspension) was added toracemic cyclopentylmandelic acid 1 (7.5 g) in methanol (20 ml) at roomtemperature. The reaction solution was allowed to stand overnight. Thecrystals were filtered and crystallized again with hot methanol. Thesecond crop of crystals was collected by filtration and treated withsodium hydroxide solution. The basic solution was extracted withmethylene chloride twice (methylene chloride solution discarded), andthen acidified with hydrochloric acid to recover the resolvedcyclopentylmandelic acid. To this resolved acid (20.6 mg in 0.1 ml ofethyl acetate), 13 μL of (+)-α-phenylethylamine was added. Theprecipitate which formed was washed with hexane three times and driedunder vacuum. The precipitate was identified by NMR as optically purecyclopentylmandelic acid, R(−)1, (2.5 g, 33.3%). M.p.: 121-122° C.[α]^(25°) _(D)=−22.5° (c=1 g/100 ml, CHCl₃). ¹H NMR (CDCl₃, 300 MHz):1.28-1.39, 1.42-1.50, 1.51-1.61, 1.64-1.73 [8H, m, (CH₂)₄], 2.93 [1H, p,CHC(OH)], 7.25-7.28, 7.32-7.35, 7.64-7.65 (5H, m, Ph) ppm.

Methyl(−)-cyclopentylmandelate, R(−)2

To a mixture of (−)-cyclopentylmandelic acid, R(−)1, (1.83 g, 8.3 mmol)and potassium carbonate (2.87 g, 21 mmol) in DMF (21 ml), methyl iodide(3.53 g, 25 mmol) was added at room temperature. The mixture was stirredat room temperature for 2 h, and then poured into water and extractedwith hexane three times. Evaporation of dried hexane extract gave acrude product. Flash chromatography of the crude product on silica gelwith 1.5:1 hexane:methylene chloride gave pure product R(−)2 (1.90 g,95%). ¹H NMR (CDCl₃, 300 MHz): 1.32-1.36, 1.43-1.61 [8H, m, (CH₂)₄],2.90 [1H, p, CHC(OH)], 3.71 (1H, s, OH), 3.79 (3H, s, CH₃), 7.25-7.28,7.31-7.35, 7.63-7.65 (5H, m, Ph) ppm.

(R)-3-Hydroxy-N-Methyl pyrrolidine, (R)3

In a 100 ml flask, 2 g (R)-3-Hydroxy pyrrolidine, 25 ml THF, 0.49 gparaformaldehyde and 1.5 g formic acid (90%) were added. The mixture wasstirred under reflux for 5 hours (until all solid disappeared), thencooled at 0° C. and added with 10 ml of NaOH solution (10 N) to adjustthe pH to about 10. The organic layer was separated and dried overMgSO₄. After filtering the dried solution and removing the solvent(THF), an oily product (1.5 g, 92%) of (R)3 was obtained. ¹H NMR (CDCl₃,300 MHz): 1.50-1.60 (m, 1H), 1.98-2.10 (m, 1H), 2.25 (s, 3H), 2.25-2.40(m, 2H), 2.50-2.60 (m, 1H), 2.61-2.70 (m, 1H), 3.80 (brs, 1H), 4.20-4.30(m, 1H).

(S)-3-Hydroxy-N-Methylpyrrolidine, (S)3

Synthesis of (S)3 was the same as for (R)3, except that the startingmaterial was (S)-3-Hydroxy pyrrolidine. The resultant product (S)3 (1.5g, 92%) was also an oil. ¹H NMR (DMSO-D6 300 MHz): 1.50-1.60 (m, 1H),1.98-2.05 (m, 1H), 2.15 (s, 3H), 2.15-2.35 (m, 2H), 2.45-2.52 (m, 1H),2.61-2.70 (m, 1H), 4.20 (brs, 1H), 4.60-4.70 (m, 1H).

3(R)—N-Methyl-3-pyrrolidinyl-2(R)-cyclopentylmandelate, 4

A solution of R(−)2 (0.7 g, 3 mmol) and (R)3 (0.7 g, 7 mmol) in 40 ml oftoluene was heated until 20 ml of toluene had distilled. Approximately0.003 g of sodium was added, and the solution was stirred and heated for2 h as the distillation was continued. More toluene was added at such arate as to keep the reaction volume constant. Additional sodium wasadded at the end of an hour. The solution was then cooled and extractedwith 3N HCl. The acid extract was made alkaline with concentrated NaOHand extracted three times with ether. Removal of dried ether solutiongave a crude oil. Flash chromatography of the crude product on silicagel with 8:1 of EtOAc and EtOH gave an oil product of 4 (0.4 g, 44%). ¹HNMR (CDCl₃, 300 MHz): 1.28-1.37, 1.51-1.70, 1.83-1.90[8H, m, (CH₂)₄],2.27-2.40 (m, 3H), 2.52-2.55 (m, 1H), 2.64-2.72 (m, 1H), 2.74-2.81 (m,1H), 2.33 (3H, s, NCH₃), 2.93 [1H, p, CHC(OH)], 3.85 (1H, bs, OH), 5.22(m, 1H), 7.24-7.27, 7.31-7.35, 7.64-7.66 (5H, m, Ph)ppm.

3(S)—N-Methyl-3-pyrrolidinyl-2(R)-cyclopentylmandelate, 5

Synthesis of 5 was the same as for 4, except (S)3 was used instead of(R)3. The resultant product 5 (0.35 g, 39%) was also an oil. ¹H NMR(CDCl₃, 400 MHz): 1.28-1.37, 1.51-1.70, 1.75-1.82[8H, m, (CH₂)₄],2.15-2.22 (m, 1H), 2.30-2.40 (m, 2H), 2.65-2.70 (m, 1H), 2.70-2.82 (m,2H), 2.35 (3H, s, NCH₃), 2.95 [1H, p, CHC(OH)], 3.82 (1H, bs, OH), 5.22(m, 1H), 7.24-7.27, 7.31-7.35, 7.64-7.66 (5H, m, Ph)ppm.

(2R,3′R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, 6 [Compound (e)]

To Compound 4 (0.3 g, 0.98 mmol) in 12 ml of dry acetonitrile, methylbromoacetate (0.5 g, 3.2 mmol) was added at room temperature. Themixture was stirred for 6 h. Evaporation of acetonitrile gave a crudeproduct. The crude product was dissolved in a small volume of methylenechloride and then poured into 50 ml of dry ethyl ether to precipitate.This step was repeated three times to obtain the pure product 6, orCompound (e), (0.3 g, 70%) as a white powder that was a mixture of twodiastereoisomers in a NMR-estimated ratio of 2:1. ¹H NMR (CDCl₃, 400MHz): 1.30-1.37, 1.41-1.50, 1.55-1.70[8H, m, (CH₂)₄], 2.10-2.27 (m, 1H),2.79-2.95 (m, 2H), 3.05, 3.60 (2s, total 3H, N—CH3), 3.75, 3.79 (2s,total 3H, O-Me), 3.95-4.40 (m, 4H), 4.68, 5.16 (2AB, total 2H,N—CH2-COOMe), 5.52-5.58 (m, 1H), 7.23-7.29, 7.31-7.38, 7.56-7.60 (5H, m,Ph)ppm.

(2R,3′S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, 7a [Compound (f)]

To Compound 5 (0.16 g, 0.52 mmol) in 8 ml of dry acetonitrile, methylbromoacetate (0.3 g, 1.9 mmol) was added at room temperature. Followingthe same procedure for 6 [Compound (e)] the pure product 7a [Compound(f)] (0.16 g, 80%) was obtained. Compound (f) was also a white powderand a mixture of two diastereoisomers in a NMR-estimated ratio of 2:1.¹H NMR (CDCl₃, 400 MHz): 1.30-1.70[8H, m, (CH₂)₄], 1.95-2.00, 2.10-2.20(m, 1H), 2.75-2.95 (m, 2H), 3.30, 3.70 (2s, total 3H, N—CH3), 3.78, 3.82(2s, total 3H, O-Me), 4.00-4.42 (m, 4H), 4.90, 5.38 (2AB, total 2H,N—CH2-COOMe), 5.52-5.58 (m, 1H), 7.23-7.29, 7.31-7.38, 7.56-7.60 (5H, m,Ph)ppm.

(2R,3′S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, 7b [Compound (g)]

To Compound 5 (0.16 g, 0.52 mmol) in 10 ml of dry acetonitrile, ethylbromoacetate (0.32 g, 1.9 mmol) was added at room temperature. Themixture was stirred for 22 hours, and the removal of acetonitrile gave acrude product. The crude product was dissolved in small volume ofethylene chloride, and then poured into a 50 ml of dry ethyl ether toafford a precipitate. This procedure was repeated three times, and thepure product 7b, or Compound (g) (0.16 g, 80%) was obtained. Compound(g) was also a white powder and a mixture of two diastereoisomers in aNMR-estimated ratio of 2:1. ¹H NMR (CDCl₃, 400 MHz): 1.32, 1.35 (2t, 3H,CH₃CH₂), 1.40-1.50, 1.53-1.63, 1.65-1.80[8H, m, (CH₂)₄], 1.93-2.11 (m2H), 2.80-2.96 M, 2H), 3.30, 3.70 (2s, 3H, N—CH3), 4.10-4.60 (m, 6H),4.79, 5.30 (2H, 2 set of dd, CH₂CO₂), 5.53 (1H, m), 7.24-7.29,7.31-7.38, 7.56-7.60 (5H, m, Ph)ppm.

Hydrolysis of Esters

Compounds (e) and (f) were combined with equimolar ratios of 0.1 N NaOH.The mixtures were stirred at room temperature for 3 hours to obtain thecorresponding racemic zwitterionic products, 8 and 9 in aqueous solution(colorless, pH about 6.5). Compound 8 is (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt. Compound 9 is (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt.

HPLC Separations for 8a, 8b, and 9a, 9b

The solutions of 8 and 9 each contained two isomers, 8a, 8b and 9a, 9b,at a ratio of 2:1 that could be separated by HPLC. The HPLC systemconsisted of a Spectra Physics (San Jose, Calif.) SP 8810 isocraticpump, a SP 8450 UV/V is detector (wavelength set to 230 nm), a SP 4290integrator, and a Supelco Discovery RP Amide C16 column. The mobilephase consisted of acetonitrile and water at a ratio of 30:70. With 100μL injection at a flow rate of 1 mL/min, the retention times were 7.2min for 8a and 9a, and 8.5 min for 8b and 9b. The effluencecorresponding to each isomer was collected, and the solvent wasevaporated to obtain the final zwitterionic isomers, 8a, 8b, and 9a, 9bas following:

(2R,1′R, 3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 8a [Compound (dd)]: white powder ¹H NMR (CDCl₃, 300 MHz):1.30-1.65 (m, 8H), 2.02-2.12 (m, 1H), 2.20-2.60 (brs, 1H), 2.60-2.80 (m,1H), 2.82-2.92 (m, 1H), 3.30 (s, 3H), 3.55-3.65 (m, 1H), 3.72-3.82 (m,1H), 3.90-4.05 (m, 2H), 4.10-4.15 (m, 1H), 5.38-5.45 (m, 1H), 7.15-7.20(m, 1H), 7.32-7.38 (m, 2H), 7.55-7.62 (m, 2H).(2R,1′S, 3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 8b [Compound (ee)]: ¹H NMR (CDCl₃, 300 MHz): 1.30-1.75 (m,8H), 2.02-2.10 (m, 1H), 2.10-2.40 (brs, 2H), 2.70-2.80 (m, 1H),2.80-2.90 (m, 1H), 2.95 (s, 3H), 3.55-3.65 (m, 2H), 3.85-4.0 (m, 3H),4.05-4.10 (m, 1H), 5.38-5.45 (m, 1H), 7.15-7.20 (m, 1H), 7.25-7.30 (m,2H), 7.50-7.60 (m, 2H).(2R,1′R, 3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 9a [Compound (ff)]: white powder, ¹H NMR (CDCl₃, 500 MHz):1.30-1.65 (m, 8H), 2.02-2.12 (m, 1H), 2.50-2.60 (m, 1H), 2.78-2.88 (m,1H), 3.25 (s, 3H), 3.65-4.05 (m, 4H), 4.15-4.30 (brs, 2H), 5.30-5.40 (m,1H), 7.13-7.23 (m, 1H), 7.26-7.32 (m, 2H), 7.55-7.60 (m, 2H).(2R,1′S, 3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 9b [Compound (gg)]: white powder, ¹H NMR (CDCl₃, 500 MHz):1.30-1.70 (m, 8H), 1.90-1.98 (m, 1H), 2.65-2.70 (m, 1H), 2.85-2.90 (m,1H), 3.15 (s, 3H), 3.65-3.90 (m, 4H), 4.05-4.10 (M, 1H), 4.15-4.22 (brs,1H), 5.35-5.42 (m, 1H), 7.18-7.23 (m, 1H), 7.27-7.32 (m, 2H), 7.53-7.58(m, 2H).Determination of Absolute Configurations

Nuclear overhauser effect (NOE) has been used to identify the absoluteconfiguration of the product 8b. Compound was dissolved in CDCl₃, andthe 2D ¹H-¹H NOESY spectrum was taken by Mercury-300BB.

Synthesis of 2S-isomersCis-(2S,5S)-2-(tert-butyl)-5-phenyl-1,3-dioxolan-4-one, 10

S(+)-mandelic acid in hexane suspension (50 g, 328 mmol) was added withpivaldehyde (42.7 ml, 396 mmol) then trifluoromethanesulfonic acid (1.23ml, 14 mmol) at room temperature. The mixture was warmed to 36° C., andthe reaction was followed by TLC for 5 hr until no starting materialcould be detected. The mixture was then cooled to room temperature andadded with 8% aqueous NaHCO₃. The water layer was removed and theorganic layer was dried over Na₂SO₄. After filtration and removal of thesolvent, 62.17 g of the crude product was obtained. Recrystallization ofthe crude product gave 44.71 g of purecis-(2S,5S)-2-(tert-butyl)-5-phenyl-1,3-dioxolan-4-one in 88% yield as aneedle-like crystal. ¹H NMR (CDCl₃, 300 MHz): 1.08 (s. 9H), 5.24 (s,1H), 5.33 (s, 1H), 7.40-7.46 (m, 5H)ppm. ¹³C NMR (CDCl₃, 300 MHz): 23.6,34.4, 77.0, 109.3, 127.0, 128.7, 129.2, 133.4, 147.2.

Cis-(2S,5S)-2-(tert-butyl)-5-phenyl-5-cyclopentyl-1,3-dioxolan-4-one, 11

At −78° C., a lithium bis-(trimethylsilyl)amide in hexane solution (120ml, 120 mmol, 1.0M in hexane) was added to compound 10 (25 g, 113.5mmol, dissolved in 100 ml of dried THF), stirred for 1 hr, followed byaddition of cyclopentyl bromide (25 g, 168 mmol). This reaction was keptat −78° C. for 4 hr, then slowly warmed up to room temperature andcontinued for overnight. The completion of the reaction was followed byTLC. With stirring, a solution of 10% of NH₄Cl (25 ml) was added in themixture. Then, the mixture was poured into a separation funnelcontaining 10% NH₄Cl solution (200 ml). The aqueous layer was discarded,and the organic layer was dried over Na₂SO₄. The solvent was removed togive a crude product, which was then re-crystallized in hexane to give apure product, 11 (20.36 g, yield 63%, white crystal). ¹H NMR (CDCl₃, 300MHz): 1.15 (s, 9H), 1.55-1.95 (m, 8H), 2.74 (m, 1H), 5.62 (s, 1H),7.44-7.56 (m, 3H), 7.88-7.91 (m, 2H) ppm. ¹³C NMR (CDCl₃, 300 MHz):23.5, 24.5, 25.3, 26.6, 35.6, 50.9, 83.2, 110.6, 124.9, 127.5, 127.9,138.9, 173.7.

S(+)-Cyclopentylmandelic Acid, 12

To a solution ofcis-(2S,5S)-2-(tert-butyl)-5-cyclopentyl-5-phenyl-1,3-dioxolan-4-one(14.35 g, 50 mmol) in 100 ml methanol and 50 ml water, 15 g of KOH wasadded slowly. The mixture was stirred and heated (65° C.) to reflux for3-4 hr, then cooled down to the room temperature, and methanol wasremoved. To the aqueous solution, 100 ml of ethyl acetate was added,then acidified to pH 1 with 3N HCl. The mixture was poured into aseparation funnel, and the organic layer was separated. The aqueouslayer was extracted two times with ethyl acetate (50 ml). The combinedorganic layers were dried over Na₂SO₄, filtered, and the solvent wasremoved to provide 13.44 g of yellowish crude product, which wasre-crystallized to give a pure product of S(+)-cyclopentylmandelic acid,12 (6.89 g, yield 62%, white crystal). ¹H NMR (CDCl₃, 300 MHz):1.28-1.75 (m, 8H), 2.94 (m, 1H), 7.24-7.34 (m, 3H), 7.62-7.68 (m, 2H).¹³C NMR (CDCl₃, 300 MHz): 25.9, 26.3, 26.4, 26.9, 47.1, 79.2, 125.8,127.7, 128.2, 140.8, 180.9.

Methyl S(+)-cyclopentylmandelate, 13

S(+)-cyclopentylmandelic acid, 12 (5.5 g, 25 mmol), and potassiumcarbonate (8.61 g, 63 mmol) in DMF (60 ml) solution was added withmethyl iodide (10.6 g, 75 mmol). The mixture was stirred at roomtemperature for 3 hr, poured into water, and extracted with hexane forthree times. Evaporation of dried hexane extract gave a pure product ofS(+)-cyclopentylmandelate, 13 (5.85 g, 100%, clear oil). ¹H NMR (CDCl₃,300 MHz): 1.32-1.61 [8H, m, (CH₂)₄], 2.90 [1H, p, CHC(OH)], 3.76 (s,3H), 3.78 (s, 1H), 7.25-7.35 (m, 3H), 7.63-7.65 (m, 2H). ¹³C NMR (CDCl₃,300 MHz): 25.9, 26.2, 26.3, 26.8, 47.1, 53.2, 79.1, 125.8, 127.3, 128.0,141.6, 176.0.

(R)-3-Hydroxy-N-Methylpyrrolidine, (R)3

In a 100 ml flask, 4 g (R)-3-Hydroxy pyrrolidine hydrochloride salt, 50ml THF and 1.3 g NaOH were added and stirred for 20 min. Then, 1.1 gparaforaldehyde and 4.8 g formic acid (90%) were added. The mixture washeated (60° C.) and stirred at reflux for 2 hr until all soliddisappeared. The mixture was cooled to 0° C., combined with 6.5 ml of 10N NaOH solution (pH about 10), and extracted twice by ethyl ether (50ml). The combined organic layer was dried over Na₂SO₄. Evaporation ofthe dried organic layer gave a yellowish, oily product of (R)3 (3.0 g,92%). ¹H NMR (CDCl₃, 300 MHz): 1.65-1.75 (m, 1H), 2.15-2.36 (m, 2H),2.33 (s, 3H), 2.55-2.59 (m, 2H), 2.76-2.85 (m, 1H), 4.30-4.40 (m, 1H),4.8-5.10 (brs, 1H). ¹³C NMR (CDCl₃, 300 MHz): 35.4, 41.9, 54.7, 64.9,70.9.

(S)-3-Hydroxy-N-Methylpyrrolidine, (S)3

Synthesis of (S)3 was the same as for (R)3, except the starting materialwas (S)-3-Hydroxypyrrolidine hydrochloride salt. The resultant product(S)3 (3.10 g, 95%) was also an oil. ¹H NMR (CDCl₃, 300 MHz): 1.50-1.60(m, 1H), 2.05-2.30 (m, 2H), 2.28 (s, 3H), 2.40-2.50 (m, 2H), 2.70-2.80(m, 1H), 4.25-4.30 (m, 1H), 4.80 (brs, 1H). ¹³C NMR (CDCl₃, 300 MHz):35.4, 41.9, 54.7, 64.9, 70.9.

(3R)N-Methyl-3-pyrrolidinyl-(S)-cyclopentylmandelate, 14

In a 250 ml 3-neck flask equipped with Dean-Stark condenser, a mixtureof methyl S(+)-cyclopentylmandelate, 13 (2 g, 8.8 mmol),(R)-3-hydroxy-N-methylpyrrolidine, (R)-3 (2 g, 20 mmol), and 100 ml ofheptane was stirred and heated (110° C.) until 20 ml of heptane had beendistilled. The temperature was reduced to 25° C., and approximately0.003 g of sodium was added. The mixture was stirred and heated to 110°C. again for 3 hr as the distillation was continued. An additional pieceof sodium (0.002 g) was added at the 1 hr point. More heptane was addedat such a rate as to keep the reaction volume constant. The mixture wascooled to 0° C., mixed with 5 ml of water, and the organic layer wasseparated. The organic layer was extracted with 3N HCl. The acid extractwas made alkaline (pH 10) with 5N NaOH and extracted three times withether. Removal of dried ether solution (over Na₂SO₄) gave a clear, oilyproduct 14 (1.6 g, 61.5%). ¹H NMR (CDCl₃, 300 MHz): 1.28-1.80 [m, 9H],2.15-2.25 (m, 1H), 2.30-2.40 (m, 1H), 2.37 (s, 3H), 2.65-2.80 (m, 3H),2.90-3.00 (m, 1H), 3.85 (1H, brs, OH), 5.22 (m, 1H), 7.20-7.35 (m, 3H),7.64-7.70 (m, 2H). ¹³C NMR (CDCl₃, 300 MHz): 26.0, 26.4, 26.5, 26.7,32.1, 42.0, 47.1, 54.8, 62.0, 76.5, 79.1, 125.8, 127.3, 128.0, 141.7,175.3.

(3S)N-Methyl-3-pyrrolidinyl-(S)-cyclopentylmandelate, 15

Following the same procedure as for 14, except (S)-3 was used instead of(R)-3, a clear, oily product of 15 (2.33 g, 89.6%) was obtained. ¹H NMR(CDCl₃, 300 MHz): 1.24-1.70 (m, 9H), 1.80-1.88 (m, 1H), 2.25-2.40 (m,2H), 2.35 (s, 3H), 2.55-2.70 (m, 2H), 2.75-2.82 (m, 1H), 2.90-3.00 (m,1H), 3.95 (1H, bs, OH), 5.22 (m, 1H), 7.24-7.40 (m, 2H), 7.64-7.69 (m,5H). ¹³C NMR (CDCl₃, 300 MHz): 26.0, 26.3, 26.4, 26.7, 32.6, 42.0, 47.1,54.9, 61.6, 76.4, 79.2, 125.8, 127.3, 128.0, 141.7, 175.2.

(2S,3′R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, 16 [Compound (h)]

Compound 14 (0.6 g, 1.96 mmol) in 30 ml of dry acetonitrile was combinedwith methyl bromoacetate (1.0 g, 6.4 mmol) at room temperature. Themixture was stirred for 3 hr. Evaporation of acetonitrile gave a crudeproduct. The crude product was dissolved in a small volume of methylenechloride and poured into a 100 ml of dry ethyl ether to obtain aprecipitate. This procedure was repeated three times and gave compound(h) as the product (0.81 g, 89%, white powder). ¹H NMR (CDCl₃, 300 MHz):1.30-1.70 (m, 8H), 1.82-1.95 (brs, 1H), 2.10-2.20 (m, 1H), 2.75-2.90 (m,2H), 3.25, 3.60 (2s, total 3H, N—CH3), 3.75, 3.79 (2s, total 3H, O-Me),4.10-4.60 (m, 4H), 4.92, 5.35 (2AB, total 2H, N—CH2-COOMe), 5.52-5.58(m, 1H), 7.23-7.38 (m, 3H), 7.56-7.60 (m, 2H). ¹³C NMR (CDCl₃, 300 MHz):25.8, 25.9; 26.3, 26.4; 26.4, 26.5; 27.0, 27.0; 29.8, 30.1; 45.9, 46.8;50.2, 51.4; 53.2, 53.2; 62.2, 63.2; 64.6, 64.7; 69.6, 69.7; 72.8, 73.1;79.4, 79.6; 125.7, 125.7; 127.6, 127.9; 128.2, 128.4; 141.0, 141.2;165.3, 165.5; 173.9, 174.2.

(2S,3′S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl-1-methylpyrrolidiniumbromide, 17 [Compound (i)]

Following the same procedure as for Compound (h), except compound 15 wasused instead of compound 14, the product Compound (i) (0.8 g, 88%, whitepowder) was obtained. ¹H NMR (CDCl₃, 300 MHz): 1.30-1.75 (m, 8H),1.80-1.90 (brs, 1H), 2.15-2.30 (m, 1H), 2.78-2.95 (m, 2H), 3.10, 3.65(2s, total 3H, N—CH3), 3.75, 3.78 (2s, total 3H, O-Me), 4.15-4.52 (m,4H), 4.85, 5.38 (2AB, total 2H, N—CH2-COOMe), 5.50-5.58 (m, 1H),7.23-7.38 (m, 3H), 7.56-7.66 (m, 2H). ¹³C NMR (CDCl₃, 300 MHz): 25.8,25.9; 26.2, 26.3; 26.3, 26.4; 26.8, 26.9; 29.4, 29.6; 45.6, 46.9; 50.1,51.4; 53.1, 53.1; 62.2, 63.3; 64.8, 64.8; 69.5, 69.8; 72.8, 73.2; 79.4,79.6; 125.6, 125.9; 127.6, 127.9; 128.2, 128.4; 140.7, 141.1; 165.2,165.5; 173.9, 174.2.

Hydrolysis of Esters & HPLC Separations

The procedures used for obtaining the 2S-isomers 18a, 18b, 19a and 19b(white powder) were the same as for 2R-isomers 8a, 8b, 9a and 9b.

(2S,1′R, 3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 18a [Compound (hh)]: ¹H NMR (CDCl₃, 300 MHz): 1.30-1.65 (m,8H), 2.02-2.45 (m, 2H), 2.82-2.90 (m, 1H), 3.10-3.18 (m, 1H), 3.25 (s,3H), 3.50-4.05 (m, 6H), 5.34-5.40 (m, 1H), 7.23-7.38 (m, 3H), 7.50-7.68(m, 2H).(2S,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 18b [Compound (ii)]: ¹H NMR (CDCl₃, 300 MHz): 1.45-1.85 (m,9H), 2.05-2.15 (m, 1H), 2.80-2.90 (m, 1H), 3.00-3.10 (m, 1H), 3.35 (s,3H), 3.70-3.80 (m, 1H), 3.90-4.10 (m, 4H), 4.22-4.35 (m, 1H), 5.50-5.60(m, 1H), 7.36-7.55 (m, 3H), 7.72-7.80 (m, 2H).(2S,1′R, 3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 19a [Compound (jj)]: ¹H NMR (CDCl₃, 300 MHz): 1.20-1.65 (m,8H), 1.95-2.10 (m, 1H), 2.20 (brs, 1H), 2.40-2.50 (m, 1H), 2.78-2.90 (m,1H), 3.15 (s, 3H), 3.70-3.90 (m, 2H), 3.96-4.20 (m, 4H), 5.38-5.50 (m,1H), 7.20-7.38 (m, 3H), 7.55-7.65 (m, 2H).(2S,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 19b [Compound (kk)]: ¹H NMR (CDCl3, 300 MHz): 1.35-1.70 (m,8H), 2.00-2.15 (m, 1H), 2.70-2.90 (m, 2H), 3.00 (s, 3H), 3.42 (brs, 1H),3.58-3.68 (m, 2H), 3.80-3.95 (m, 3H), 4.08-4.18 (m, 1H), 5.38-5.48 (m,1H), 7.20-7.40 (m, 3H), 7.55-7.62 (m, 2H).Receptor Binding Affinity

Receptor binding studies on soft anticholinergics isomers and theirzwitterionic metabolite isomers, as well as glycopyrrolate, andN-methylscopolamine were performed with N—[³H]-methyl-scopolamine (NMS)in assay buffer (phosphate-buffered saline, PBS, without Ca⁺⁺ or Mg⁺⁺,pH 7.4), following the protocol from Applied Cell Science Inc.(Rockville, Md.). A 10 mM NaF solution was added to the buffer as anesterase inhibitor. The assay mixture (0.2 mL) contained 20 μL dilutedreceptor membranes (receptor proteins: M₁, 38 μg/mL; M₂, 55 μg/mL; M₃,27 μg/mL; M₄, 84 μg/mL). The final concentration of NMS for the bindingstudies was 0.5 nM. Specific binding was defined as the difference in[³H]NMS binding in the absence and presence of 5 μM atropine for M₁ andM₂ or 1 μM atropine for M₃ and M₄. Incubation was carried out at roomtemperature for 2 hr. The assay was terminated by filtration through aWhatman GF/C filter (presoaked overnight with 0.5% polyethyleneimine).The filter was then washed six times with 1 mL ice cold buffer (50 mMTris-HCl, pH 7.8, 0.9% NaCl), transferred to vials, and 5 mL ofScintiverse was added. Detection was performed on a Packard 31800 liquidscintillation analyzer (Packard Instrument Inc., Downer Grove, Ill.).Data obtained from the binding experiments were fitted to the equation%[³H] NMS bound=100−[100x^(n)/k/(1+x^(n)/k)], to obtain the Hillcoefficient n, and then to the equation %[³H] NMSbound=100−[100x^(n)/IC₅₀/(1+x^(n)/IC₅₀)], to obtain the IC₅₀ values (xbeing the concentration of the tested compound). Based on the method ofCheng and Prusoff, K_(i) was derived from the equationK_(i)=IC₅₀/(1+L/K_(d)), where L is the concentration of the radioligand.IC₅₀ represents the concentration of the drug causing 50% inhibition ofspecific radioligand binding, and K_(d) represents the dissociationconstant of the radioligand receptor complex. Data were analyzed by anon-linear least-square curve-fitting procedure using Scientist software(MicroMath Inc., Salt Lake City, Utah).

Determination of pA₂ Values

Male guinea pigs weighing about 400 g were obtained from Harlan Inc.(Indianapolis, Ind.) and fasted overnight. Animals were sacrificed bydecapitation, and the ileum (the region of 5 cm upward of the cecum) wasisolated and removed. The ileum was cut into 2.5 cm pieces and suspendedin an organ bath containing 30 mL of mixture of Tyrode's solution and0.1 mM hexamethonium bromide. The organ bath was constantly aerated withoxygen and kept at 37° C. One end of the ileum strip was attached to afixed support at the bottom of the organ bath, and the other end to anisometric force transducer (Model TRN001, Kent Scientific Corp., Conn.)operated at 2-10 g range. The ileum strip was kept at a 2 g tension, andcarbachol was used as antagonist. The ileum contracted cumulatively uponthe addition of consecutive doses of carbachol (10-20 μL of2×10⁻⁴-2×10⁻³M in water solution). Contractions were recorded on aphysiograph (Kipp & Zonen Flarbed Recorder, Holland). After the maximumresponse was achieved, the ileum was washed three times, and a freshTyrode's solution containing appropriate concentration of the antagonist(anticholinergic compound tested) was replaced. An equilibration time of10 min was allowed for the antagonists before the addition of carbachol.In each experiment, 5 to 6 different concentrations were used, and aSchild plot was used to obtain the pA₂ values. Four trials wereperformed for each antagonist.

In Vivo Mydriatic Studies

The mydriatic effects of eight completely resolved zwitterionic isomerswere compared to those of glycopyrrolate, tropicamide, (±)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt [(±)-GA or Compound (bb)] and (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt [(2R)-GA or Compound (cc)] in rabbit eyes. Four healthy, maleNew-Zealand white rabbits weighting about 3.5 kg were used. 100 μL ofcompound in water solution (pH 6.5) at various concentrations wereadministered in the eyes. Compound solutions were applied to one eye,and water was applied to the other eye that served as control.Experiments were carried out in a light- and temperature-controlledroom. At appropriate time intervals, the pupil diameters of both eyeswere recorded. Percent difference in pupil diameters between eachtime-point and zero time-point were calculated for both treated andcontrol eyes and reported as mydriatic responses. Control eye dilationswere monitored to determine whether systemic absorption had occurred ornot. The area under the mydriatic response-time curve (AUC^(eff)) wascalculated by the trapezoidal rule, and it was used to compare theactivity and duration of action of the tested compounds.

Statistical Analysis

Receptor binding affinities and pA₂ values were compared using studentt-tests. Mydriatic activities (maximum response Rmax % and area underthe effect curves AUC^(eff)) were compared using ANOVA. A significancelevel of P<0.05 was used in all cases.

Results and Discussion

Synthesis

Five soft anticholinergic ester isomers and eight zwitterionicmetabolite acid isomers were newly synthesized. The 2R diastereoisomers[Compounds (e), (f), (g), (dd), (ee), (ff) and (gg)] were obtained bythe synthetic pathways described below.

As shown in Scheme 1, first the racemic cyclopentylmandelic acid 1 wassynthesized with cyclopentylmagnesium bromide and benzoylformic acid.This racemic acid was resolved by repeated crystallization of the saltsproduced between this acid and (−)-strychnine. The left rotatory(−22.5°) optically pure free acid R(−)1 was recovered by basification ofthe salts with sodium hydroxide solution followed by acidification withhydrochloric acid. Methylation of R(−)1 with methyl iodide and potassiumcarbonate in DMF at room temperature yields methyl2R(−)cyclopentylmandelate, R(−)2. Transesterfication of R(−)2 withR-3-hydroxy-N-methylpyrrolidine, (R)-3 (made from R-3-hydroxypyrrolidinewith paraformaldehyde and formic acid), gave(3R)—N-methyl-3-pyrrolidinyl-2R-cyclopentylmandelate 4; or withS-3-hydroxy-N-methylpyrrolidine (S)-3 (made from S-3-hydroxypyrrolidinewith paraformaldehyde and formic acid), gave(3S)—N-methyl-3-pyrrolidinyl-2R-cyclopentyl mandelate 5. Quaternizationof 4 and 5 with methyl or ethyl bromoacetate in acetonitrile gave 6[Compound (e)], 7a [Compound (f)], and 7b [Compound (g)]. Each of thesehas two diastereoisomers, due to the nitrogen chiral center, with aratio of 2 to 1 (R:S=2:1) that was shown in ¹H NMR spectra. Hydrolysisof 6 [Compound (e)] and 7a [Compound (f)] gave their zwitterionic innersalts 8 and 9. Each zwitterionic salt also possesses twodiastereoisomers with a ratio of 2 to 1 that could be separated by HPLCto give zwitterionic isomers 8a, 8b & 9a and 9b. From ¹H NMR, 8a, 8b,and 9a, 9b were evidenced to be pairs of diastereoisomers based onchiral nitrogen. To identify the absolute configuration of theseisomers, 8b was chosen and dissolved in CDCl₃ for the investigation ofnuclear overhauser effect (NOE). The 2D ¹H-¹H NOESY spectrum showed thatthe methyl group on the nitrogen was at the same side as the hydrogen atthe 3-position of pyrrolidinium ring. Accordingly, the configuration ofthe nitrogen should be the S form, and the absolute stereochemistry of8b was proved to be (2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt [Compound (ee)]. Therefore, 8a was (2R,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt [Compound (dd)]; 9a was (2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt [Compound (ff)]; and 9b was (2R,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt [Compound (gg)].

Grover and coworkers previously reported [J. Org. Chem. 65: 6283-6287(2000)] the highly stereoselective synthesis of (S)-cyclopentylmandelicacid in five steps starting with (S)-mandelic acid. Modification oftheir procedure afforded pure S(+)-cyclopentylmandelic acid in threesteps with good yield. As depicted in Scheme 2, reaction ofS(+)-mandelic acid with pivaldehyde in the presence of the catalysttrifluoromethanesulfonic acid gave the product ofcis-(2S,5S)-2-(tert-butyl)-5-phenyl-1,3-dioxolan-4-one, 10, in about 90%yield. At −78° C., deprotonation of 10 with lithiumbis(trimethylsilyl)amide followed by adding cyclopentyl bromidegeneratedcis-(2S,5S)-2-(tert-butyl)-5-cyclopentyl-5-phenyl-1,3-dioxolan-4-one,11. Base hydrolysis of 11 with potassium hydroxide, followed byacidification with hydrochloric acid provided the expected(S)-(+)-cyclopentylmandelic acid 12. After this step, the sameprocedures as for 8a, 8b, 9a and 9b including methylation,esterification, quaternization and hydrolyses were followed to give thefinal four zwitterionic isomers, (2S,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt 18a [2S1′R3′R-GA or Compound (hh)]; (2S,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt 18b [2S1′S3′R-GA or Compound (ii)]; (2S,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt 19a [2S1′R3′S-GA or Compound (jj)]; and (2S,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt 19b [2S1′S3′S-GA or Compound (kk)]. They were alsocharacterized by NMR.

Receptor Binding Studies

The receptor binding affinities of soft analogs, pK_(i), determined byradioligand binding assays using human cloned muscarinic receptorsubtypes, M₁-M₄, are presented in Table 6.

TABLE 6 Receptor binding affinities, M₃/M₂ selectivities, and pA₂values. Subtypes of cloned muscarinic receptors^(a) Selectivity^(b)Compound M₁ M₂ M₃ M₄ M₃/M₂ pA₂ ^(c) (a)^(d) 7.91 ± 0.05 7.79 ± 0.11 7.80± 0.10 8.29 ± 0.19 1.0 ± 0.0 7.90 ± 0.13 (1.02 ± 0.12) (1.25 ± 0.08)(1.17 ± 0.18) (1.12 ± 0.05) (b)^(d) 7.51 ± 0.17 7.32 ± 0.07 7.54 ± 0.157.94 ± 0.09 1.8 ± 0.7 7.36 ± 0.34 (0.91 ± 0.09) (1.23 ± 0.06) (1.18 ±0.08) (1.18 ± 0.09) (bb)^(d) 6.19 ± 0.06 5.48 ± 0.13 5.84 ± 0.07 6.44 ±0.06 2.4 ± 0.7 6.42 ± 0.30 (1.11 ± 0.06) (1.02 ± 0.20) (1.01 ± 0.07)(0.84 ± 0.06) (c)^(e) 8.89 ± 0.04 8.87 ± 0.05 9.00 ± 0.06 9.52 ± 0.011.4 ± 0.2 8.31 ± 0.05 (0.83 ± 0.11) (1.10 ± 0.11) (0.83 ± 0.01) (0.83 ±0.01) (d)^(e) 8.67 ± 0.16 8.84 ± 0.34 8.74 ± 0.02 8.85 ± 0.13 1.1 ± 1.18.55 ± 0.16 (0.86 ± 0.08) (0.92 ± 0.01) (1.09 ± 0.15) (0.89 ± 0.02)(cc)^(e) 8.11 ± 0.16 7.48 ± 0.12 8.12 ± 0.10 8.23 ± 0.12 4.4 ± 0.3 7.20± 0.19 (1.12 ± 0.25) (0.95 ± 0.11) (0.80 ± 0.01) (1.02 ± 0.10) (e)^(f)8.99 ± 0.04 9.01 ± 0.06 9.06 ± 0.14 9.45 ± 0.01 1.1 ± 0.1 — (1.19 ±0.12) (1.03 ± 0.09) (1.03 ± 0.18) (1.52 ± 0.66) (f)^(f) 8.50 ± 0.03 7.90± 0.04 8.60 ± 0.09 8.87 ± 0.09 5.0 ± 1.1  —^(h) (1.30 ± 0.20) (1.07 ±0.17) (1.04 ± 0.27) (1.08 ± 0.01) (h)^(f) 7.23 ± 0.01 7.22 ± 0.03 6.99 ±0.08 7.57 ± 0.01 0.6 ± 0.1 — (0.98 ± 0.06) (1.09 ± 0.18) (1.15 ± 0.13)(1.11 ± 0.03) (i)^(f) 6.40 ± 0.05 6.47 ± 0.08 5.95 ± 0.02 6.39 ± 0.010.3 ± 0.0 — (0.92 ± 0.09) (0.99 ± 0.16) (1.06 ± 0.03) (1.44 ± 0.75)(j)^(f) 8.68 ± 0.11 8.21 ± 0.10 8.64 ± 0.07 8.71 ± 0.38 2.8 ± 0.8 —(1.21 ± 0.33) (1.27 ± 0.11) (1.33 ± 0.16) (1.15 ± 0.03) (dd)^(g) 7.04 ±0.09 6.43 ± 0.07 6.95 ± 0.04 7.00 ± 0.05 3.5 ± 0.2 6.32 ± 0.23 (0.97 ±0.13) (0.85 ± 0.21) (1.06 ± 0.04) (0.93 ± 0.01) (ee)^(g) 8.13 ± 0.067.63 ± 0.02 8.15 ± 0.02 8.33 ± 0.04 3.3 ± 0.0 7.45 ± 0.21 (1.25 ± 0.01)(0.82 ± 0.15) (0.84 ± 0.17) (1.00 ± 0.06) (ff)^(g) 7.98 ± 0.01 7.39 ±0.09 8.04 ± 0.01 8.15 ± 0.06 5.2 ± 0.7 7.33 ± 0.28 (1.02 ± 0.03) (0.80 ±0.22) (0.96 ± 0.03) (1.01 ± 0.06) (gg)^(g) 8.32 ± 0.04 7.64 ± 0.01 8.46± 0.12 8.56 ± 0.07 5.5 ± 1.1 7.15 ± 0.12 (1.01 ± 0.01) (1.00 ± 0.04)(0.80 ± 0.21) (0.86 ± 0.06) (hh)^(g) 5.87 ± 0.04 5.65 ± 0.06 5.54 ± 0.165.79 ± 0.12 0.8 ± 0.1 5.14 ± 0.38 (1.06 ± 0.05) (1.24 ± 0.07) (1.02 ±0.12) (0.88 ± 0.04) (ii)^(g) 6.67 ± 0.06 6.35 ± 0.01 6.22 ± 0.05 6.47 ±0.01 0.7 ± 0.0 5.69 ± 0.13 (1.08 ± 0.03) (1.01 ± 0.01) (1.04 ± 0.08)(1.30 ± 0.28) (jj)^(g) <4.5 <4.5 <4.5 <4.5 — <4 — — — — (kk)^(g) 5.84 ±0.06 5.61 ± 0.00 5.61 ± 0.09 5.85 ± 0.05 1.0 ± 0.2 5.03 ± 0.26 (1.13 ±0.10) (1.20 ± 0.02) (1.03 ± 0.01) (0.95 ± 0.18) glycopyrrolate 9.76 ±0.05 9.19 ± 0.18 8.73 ± 0.05 9.90 ± 0.08 0.4 ± 0.2 8.57 ± 0.12 (1.37 ±0.20) (0.99 ± 0.11) (1.14 ± 0.25) (1.02 ± 0.01) scopolamine 9.69 ± 0.019.18 ± 0.21 9.29 ± 0.12 9.92 ± 0.21 1.3 ± 0.4 9.16 ± 0.19 methyl bromide(0.92 ± 0.10) (1.02 ± 0.02) (1.07 ± 0.01) (0.90 ± 0.04) ^(a)Receptorbinding at cloned human muscarinic receptors (M₁-M₄ subtypes); pK_(i)data represent mean ± SD of 3 experiments, and the numbers inparentheses denote Hill slopes. ^(b)M₃/M₂ affinity ratio (times) ^(c)pA₂values were determined on 4-6 ileum strips obtained from differentanimals, and data represent mean ± SD. ^(d)Racemic forms. ^(e)Isomersbased on the chiral center 2. ^(f)Isomers based on the chiral centers 2& 3′. ^(g)Isomers based on the chiral centers 2, 3′, & 1′. ^(h)Data notavailable or not detectable.

The pK_(i) of newly synthesized isomers were compared with that of theracemic and 2R isomeric parent soft drugs [the methyl ester Compound (c)and the ethyl ester Compound (d)], racemic and 2R isomeric GA (thezwitterionic metabolite), i.e., Compounds (bb) and (cc), as well asthose of glycopyrrolate and N-methylscopolamine. pK_(i) of the racemicforms, Compound (a) and Compound (b), showed lower receptor bindingaffinities than their corresponding 2R isomers (7.8-8.3 vs. 8.7-9.5),confirming that stereospecificity is important at these receptors. Thepotencies of these 2R isomers are similar to those of glycopyrrolate(8.7-9.9) and N-methylscopolamine (9.2-9.9). Resolution of 2 and 3′chiral centers of racemic Compound (a) resulted in four stereoisomers,Compounds (e), (f), (h) and (i) with pK_(i) values of 9.0-9.5, 7.9-8.9,7.0-7.6 and 6.0-6.5, respectively. These numbers indicate that among themethyl ester isomers, not only 2R isomers are more potent than thecorresponding 2S isomers, but also that 3′R isomers are more potent than3′S isomers. The 2R3′S isomer of the ethyl ester, Compound (j), showed apK_(i) value of 8.2-8.7, the same as the 2R3′S isomer of the methylester. In the same table, the M₃/M₂ muscarinic-receptorsubtype-selectivities were also calculated. Contrary to the previouslyreported 2R isomer of the methyl and ethyl esters, Compounds (c) and(d), that show no M₃/M₂ subtype selectivity, the 2R3′S isomers of themethyl and ethyl esters, Compounds (e) and (j), show significantlyincreased M₃/M₂ muscarinic-receptor subtype-selectivity (p<0.01, t-testassuming equal variances). The M₃ affinity was 5.0±1.1 times of M₂affinity in the case of Compound (e), and 2.8±0.8 times in the case ofCompound (j). This indicates that the configuration of chiral center 3′may play an important role in the safety profile of this type of softanticholinergics.

The receptor-binding pK_(i) of racemic (±) GA, i.e., Compound (bb), andisomeric 2R-GA, i.e., Compound (cc), obtained earlier are also shown inTable 6. In agreement with soft drug design principles that the acidicmoiety formed by hydrolysis of the parent soft drug ester inactivatesthe drug, the zwitterions were found considerably less active than theircorresponding parent esters, e.g. pK_(i) of (±)GA or Compound (bb),5.5-6.4, vs. Compound (a), 7.8-8.3, and Compound (b), 7.3-7.9; andpK_(i) of 2R-GA or Compound (cc), 7.5-8.2, vs. Compound (c), 8.9-9.5,and Compound (d), 8.7-8.9 (3-4). As discussed previously, thezwitterionic metabolite retains some activity because the electronicdistribution in its structures somewhat resembles those of the neutral,active anticholinergics. In this study, to obtain a better picture ofthe stereospecificity/stereoselectivity of this type of anticholinergic,the zwitterionic form was chosen as a model compound for theinvestigation, since the zwitterion GA, either in its racemic or its 2Risomeric form, was very soluble and stable in aqueous solutions (bufferor biological media, pH 6-8). In addition, 2R-GA [Compound (cc)] hasbeen found active at topical sites (e.g. in rabbit eyes), and could beexcreted unchanged, rapidly through urine (t_(1/2) 10-15 min after i.v.in rats). In Table 6, the pK_(i) of the completely resolved eightisomers of ±GA [Compound (bb)], 2R1′R3′R-GA [Compound (dd)], 2R1′S3′R-GA[Compound (ee)], 2R1′R3′S-GA [Compound (ff)], 2R1′S3′S-GA [Compound(gg)], 2S1′R3′R-GA [Compound (hh)], 2S1′S3′R-GA [Compound (ii)],2S1′R3′S-GA [Compound (jj)], 2S1′S3′S-GA [Compound (kk)] was in a widerange of 4.5-8.6. In all cases, the 2R isomers are more potent than the2S isomers, and the 1′S isomers are more potent than the 1′R isomers.The comparative potencies for 3′R and 3′S isomers varied depending onthe configuration of chiral center 2, e.g. 2R1′R3′S>2R1′R3′R and2R1′S3′S>2R1′S3′R; but 2S1′R3′R>2S1′R3′S and 2S1′S3′R>2S1′S3′S. Also,the same as previous methyl ester isomers, among 2R isomers of the acid,the 2R3′S isomers (2R1′R3′S and 2R1′S3′S) showed highest M₃/M₂muscarinic-receptor subtype-selectivities (5.2-5.5 times) followed bythe 2R3′R isomers (2R1′R3′R and 2R1′S3′R, 3.3-3.5 times). The 2S isomersdid not show any M₃/M₂ selectivity. Thus, the importance of the chiralcenter 2 and 3′ configuration (2R3′S) on the M₃/M₂ selectivity of thistype of anticholinergics has been demonstrated.

In order to show the comparative stereoselectivity (times) based on eachchiral center, the ratio of binding activities of each correspondingpaired isomers was calculated, and the results are shown in Table 7. Theresults displayed are comparative potencies (times) calculated from thereceptor binding affinities, pK_(i), in Table 6. The difference inreceptor binding affinities between 2R and 2S isomers is significant (27to 447 times for the methyl ester isomers, and 6 to 4467 times forzwitterion isomers). The 3′R isomers of the methyl ester (with chiralcenter 1 unresolved, 2R3′R & 2S3′R methyl esters) are more active (1.5to 12.9 times) than their corresponding 3′S isomers (2R3′S & 2S3′Smethyl esters). However, in the acid, the 3′S isomers were not alwaysmore active than the corresponding 3′R isomers, e.g. in 2R isomers,3′S>3′R (2R1′R3′S>2R1′R3′R and 2R1′S3′S>2R1′S3′R); but in 2S isomers,3′R>3′S (2S1′R3′R>2S1′R3′S and 2S1′S3′R>2S1′S3′S). Also, there are moresignificant differences between 2R1′R3S′ and 2R1′R3′R than between2R1′S3′S and 2R1′S3′R (8.7 to 14.1 times vs. 1.0 to 2.0 times), andbetween 2S1′R3′R and 2S1′R3′S than between 2S1′S3′R and 2S1′S3′S (1.1.0to 23.4 times vs. 4.1 to 6.8 times). These results indicate that theactivity based on chiral center 3′ can be affected by the configurationof the other two chiral centers, 2 and 1′. When comparing all eightzwitterion isomers (with all three chiral centers resolved), it clearlyshows that 1′S isomers were more active than the corresponding 1′Risomers in all cases (1.8-22.4 times).

TABLE 7 Comparative stereoselectivities^(a) Subtypes of clonedmuscarinic receptors^(b) Compound M₁ M₂ M₃ M₄ Description^(f) MethylEsters 2R3′S/2S3′S^(c) 125.9 26.9 446.7 302.0 2R > 2S 2R3′R/2S3′R^(c)57.5 61.7 117.5 75.9 2R3′R/2R3′S^(d) 3.1 12.9 2.9 3.8 3R > 3S2S3′R/2S3′S^(d) 6.8 5.6 11.0 1.5 Zwitterions 2R1′R3′R/2S1′R3′R^(c) 14.86.0 25.7 16.2 2R >> 2S 2R1′S3′R/2S1′S3′R^(c) 28.8 19.1 85.1 72.42R1′R3′S/2S1′R3′S^(c) 3020.0 776.2 3467.4 4466.8 2R1′S3′S/2S1′S3′S^(c)302.0 107.2 707.9 512.9 2R1′R3′S/2R1′R3′R^(d) 8.7 9.1 12.3 14.1 3S > 3R2R1′S3′S/2R1′S3′R^(d) 1.5 1.0 2.0 1.7 2S1′R3′R/2S1′R3′S^(d) 23.4 14.111.0 19.5 3R > 3S 2S1′S3′R/2S1′S3′S^(d) 6.8 5.5 4.1 4.22R1′S3′R/2R1′R3′R^(e) 12.3 15.8 15.8 21.4 1R < 1S 2R1′S3′S/2R1′R3′S^(e)2.2 1.8 2.6 2.6 2S1′S3′R/2S1′R3′R^(e) 6.3 5.0 5.2 4.82S1′S3′S/2S1′R3′S^(e) 21.9 12.9 12.9 22.4 ^(a)Affiinty ratio (times)between each two isomers based on each of the three different chiralcenters. ^(b)Receptor binding at cloned human muscarinic receptors(M₁-M₄ subtypes) ^(c)Affinity ratio based on the chiral center 2.^(d)Affinity ratio based on the chiral center 3. ^(e)Affinity ratiobased on the chiral center 1. ^(f)Concluded stereoselectivities

In all cases, the Hill coefficients (n) were not very different fromunity indicating that, in general, drug-receptor interactions obeyed thelaw of action and binding took place at only one site.

pA₂ Studies

The pA₂ values determined from guinea pig ileum contraction assays,which represent the negative logarithm of the molar concentration of theantagonist that produces a two-fold shift to the right in an agonist'sconcentration-response curve, are a classical functional study ofanticholinergic affinity (at M₃ muscarinic receptors). For the softanticholinergics of the present study, the pA₂ values obtained fromileum longitudinal contractions by using carbachol as agonist with themethod of van Rossum [Arch. Int. Pharcodyn. 143: 299-330 (1963)] arepresented in Table 6. The pA₂ values are in general, comparable to thepK_(i) values obtained in the M₃ receptor binding studies. The pA₂values of newly developed zwitterionic isomers significantly differedbetween 2R and 2S configurations (6.32 to 7.45 and <4 to 5.69,respectively, p<0.01, t-test assuming equal variances). Similar to theabove reported 2R isomer (2R-GA), the pA₂ values of completely resolved2R isomers (2R1′R3′R-GA, 2R1′S3′R-GA, 2R1′R3′S-GA, and 2R1′S3′S-GA) are1 to 2 less than those of the corresponding 2R ethyl and methyl parentester soft drugs, indicating a one to two order of magnitude lessactivity of these zwitterionic compounds. The retained moderate activityof some zwitterionic metabolite isomers is probably due to aspatially-close structures that resembles those of the neutral, activeanticholinergics. In the active 2R isomers, while 2R1′R3′R-GA showed alower value (6.32), all others showed a similar moderate contractionactivity (about 7.15 to 7.45).

Mydriatic Activities

The mydriatic effects of the fully resolved eight zwitterionic isomerswere compared to those of (±)GA, 2R-GA, glycopyrrolate and tropicamidein vivo in rabbits. Following a 100 μl topical administration, themydriatic responses were recorded at appropriate time-intervals as %changes in pupil size. The maximum response (R_(max), % change in pupilsize at 30 min to 1 h after administration) and area under theresponse-time curve (AUC^(eff) ₀₋₁₆₈h) are shown in Table 8.

TABLE 8 Maximum response (R_(max), maximum % change in pupil size) andarea under the response-time curve (AUC_(eff)) after topicaladministration (0.1 mL).^(a) Compound Conc. (%) R_(max) (%) AUC^(eff)_(0-168 h) (±)GA^(b) [Cpd (bb)] 0.01 1.85 ± 2.14 0.7 ± 0.9 1 45.37 ±8.19  119 ± 34  2R-GA^(b) [Cpd (cc)] 0.01 31.00 ± 7.14  73 ± 24 0.150.34 ± 7.92  182 ± 40  2R1′R3′R-GA [Cpd (dd)] 0.1 24.40 ± 8.33  89 ± 502R1′S3′R-GA [Cpd (ee)] 0.1 51.79 ± 16.62 308 ± 106 2R1′R3′S-GA [Cpd(ff)] 0.1 43.90 ± 7.63  216 ± 29  2R1′S3′S-GA [Cpd (gg)] 0.1 47.32 ±19.64 274 ± 134 2S1′R3′R-GA [Cpd (hh)] 0.1 0.00 ± 0.00 0 ± 0 0.4 7.44 ±0.60 11 ± 1  2S1′S3′R-GA [Cpd (ii)] 0.1 3.87 ± 4.49 13 ± 15 0.4 14.88 ±1.19  37 ± 3  2S1′R3′S-GA [Cpd (jj)] 0.1 0.00 ± 0.00 0 ± 0 0.4 0.00 ±0.00 0 ± 0 2S1′S3′S-GA [Cpd (kk)] 0.1 3.87 ± 4.49 13 ± 15 0.4 11.01 ±3.81  28 ± 2  glycopyrrolate^(b) 0.05 48.73 ± 12.66 2476 ± 847  0.152.95 ± 10.93 3732 ± 866  tropicamide^(b) 0.5 44.64 ± 11.17 451 ± 121^(a)Data represent mean ± SD of four trials. ^(b)Data adapted from othertesting.

The results indicate that, as in the in vitro studies, the 2R isomersare much more potent than the 2S isomers (even when the 2S dose wasincreased to 0.4%); and 2R1′R3′R-GA is less potent than the other three2R isomers. In FIG. 6, the activity-time profiles of four 2R and one 1′Sisomers (the most active S isomer) at 0.1% are displayed. Thepupil-dilating potency of the most potent three 2R isomers at a dose of0.1% is similar to that of 0.05 to 0.1% of glycopyrrolate and 0.5% oftropicamide, however, their duration of actions was much shorter thanthat of the “hard” glycopyrrolate (AUC 200-300 vs. 2500, respectively),and somewhat shorter than that of tropicamide, in agreement with softdrug design principles. The activities of 2R1′S3′R-GA (the most activezwitterionic isomer) and glycopyrrolate lasted for 10 h and 144 h,respectively, as displayed in FIG. 7. These results indicate that a goodpharmacological effect can be achieved by some 2R zwitterionic isomers,and these isomers can be rapidly eliminated from the body. Furthermore,the active 2R zwitterionic isomers did not cause any observableirritation reactions, such as eye-closing, lacrimation, mucous dischargeas well as change in the intraocular pressure during the topicalapplications; and unlike other conventional anticholinergics, these 2Rzwitterionic isomers did not induce dilation of the pupil in thecontralateral (water-treated) eyes, indicating no or low systemicside-effects. Therefore, these soft drugs are safe, promising shortacting anticholinergics with the possibility of largely reduced unwantedside effects.

CONCLUSION

Isomers of N-substituted soft anticholinergics based on glycopyrrolate,the methyl and ethyl esters, and their zwitterionic metabolite weresynthesized and separated. Their pharmacological activities wereevaluated in vitro and in vivo. The receptor binding (pK_(i)) resultsindicate that stereo-specificity and stereo-selectivity are veryimportant in these soft anticholinergics. There were three chiralcenters presented in the structure of these compounds. The mostsignificant improvement of the receptor binding activity was observed in2R configuration, followed by 1′S. The activities of 3′R and 3′S couldbe affected by the configurations of the other two chiral centers. Theimprovement of M₃/M₂ muscarinic-receptor subtype-selectivity was foundmost significant in 2R3′S configurations followed by 2R3′R. Theconfiguration of chiral center 1′ showed no effect on M₃/M₂muscarinic-receptor subtype-selectivity. Comparable results obtainedfrom guinea pig ileum assays (pA₂), and rabbit mydriasis test onzwitterionic isomers further confirmed the stereo-specificity of theseanticholinergics. The pharmacological potency of eight zwitterionicisomers was determined to be2R1′S3′S=2R1′S3′R=2R1′R3′S>2R1′R3′R>2S1′S3′R>2S1′S3′S=2S1′R3′R>2S1′R3′S(student t-test, p<0.05). When topically administered (0.1%) in rabbiteyes, some 2R-zwitterion isomers (2R1′S3′S, 2R1′S3′R and 2R1′R3′S)showed similar mydriatic potencies to that of glycopyrrolate andtropicamide, however, their mydriatic effects were of considerablyshorter duration, and they did not induce dilation of the pupil in thecontralateral, water-treated eyes, indicating that, in agreement withtheir soft nature, they are locally active, but safe and have a lowpotential to cause systemic side effects. The usefulness and safety ofthese glycopyrrolate-based soft anticholinergics have been thereforefurther proved.

Synthesis and Biological Testing of Tiotropium Derivatives

Structure of the Compounds

b) Synthesis of a New Tiotropium Analog

By following the inactive metabolite approach, a metabolically sensitiveester function was introduced into the tiotropium molecule resulting ina new soft anticholinergic analog. Intermediate XIII was prepared by theknown Grignard reaction of dimethyl oxalate with 2-thienylmagnesiumbromide (XII, see Scheme 3 below). Compound XIII was then submitted toknown transesterification with scopine (XIV) catalyzed by sodium metalgiving the corresponding ester XV. Finally, quaternization with ethylbromoacetate gave the target compound II (R=Et).

2-Thienylmagnesium bromide was prepared in the usual manner frommagnesium and 2-bromothiophene in ether. Scopine (XIV) was obtained fromscopolamine hydrobromide by treatment with sodium borohydride in ethanolin moderate yield as described in GB 1,469,781 (1974).

The above synthesis of the soft tiotropium bromide analog leads to asingle isomer in a yield of 70%. Tlc indicated no further new componentin the reaction mixture of the final quaternization step and it could beshown by NMR (two dimensional ROESY technique) that theethoxycarbonylmethyl group is in proximity to the oxirane ring. Thisfinding is somewhat surprising as the other isomer with N-methylpointing toward the oxirane ring would be sterically less crowded.

Stereochemistry of Compound (w) obtained by two-dimensional ROESYexperiment:

The numbers denote ¹H chemical shifts and coupling constants, the doublearrows indicate steric proximities.

Preparation of 6β,7β-epoxy-3β-hydroxy-8-ethoxycarbonylmethyl-8-methyl-1αH,5αH-tropaniumbromide, di-2-thienylglycolate, Compound (w)

The scopine ester, represented by formula XV in Scheme 3 above, wasprepared as described above, or by conventional methods as described inEP418716 (equivalent to U.S. Pat. No. 5,610,163). Then the scopine ester(70 mg, 0.18 ml) is dissolved in 2 ml of acetone. Ethyl bromoacetate(150 microliters, 0.45 mM) is added and the mixture is allowed to reactat 20° C. for eight days. The solvent is evaporated in vacuo, 8 ml ofwater is added and the organic material is extracted with chloroform.The desired quaternary salt is in the aqueous phase and is obtained bylyophilization. Yield 70 mg (70%). Melting point: 115° C. Thin layerchromatography on Al₂O₃: R_(f)=0.3 (CHCl₃—CH₃OH, 4:1) (3 times 4 ml).The product, Compound (w), has the structural formula II shown in Scheme3 above.

Preparation of6β,7β-epoxy-3β-hydroxy-8-methyl-8-(2,2,2-trichloroethoxycarbonylmethyl)-1αH,5αH-tropanium bromide, di-2-thienylglycolate, Compound (x)

To the scopine ester XV (0.5 mM) in 3 ml of anhydrous acetonitrile, 1.5mM of trichloroethyl bromoacetate was added. The mixture was stirredunder argon for three days and the acetonitrile was removed underreduced pressure. To the oily residue, 15 ml of water was added andextracted with chloroform (3 times 5 ml). The aqueous solution waslyophilized to give the product as a white solid. Yield: 257 mg (79%).Melting point 105° C., R_(f)=0.65 (CHCl₃—CH₃OH, 4:1). The product hasthe structural formula:

Preparation of6β7β-epoxy-3β-hydroxy-8-carboxymethyl-8-methyl-1αH,5αH-tropanium,di-2-thienylglycolate inner salt, Compound (aa)

A suspension of 0.35 mM Compound (x) and Zn dust (0.6 mM) in acetic acid(1.5 ml) was stirred for 3 hours. To that mixture, water (2 ml) andchloroform (2 ml) were added and filtered. The solvents were evaporatedin vacuo, 3 ml of water was added and the solution was lyophilized. Thecrude product was dissolved in methanol (2 ml) and purified bychromatography on Sephadex LH-20. The resulting oil was dissolved inmethanol (2 ml) and precipitated with ethyl acetate (1 ml) to give thesolid product, Compound (aa). Yield 67 mg (38%), melting point 158-165°C. (decomp). R_(f)=0.45 (CHCl₃—CH₃OH 4:1) on aluminum oxide. The producthas the structural formula:

Pharmacology1: Receptor Binding Assay

Evaluation of the affinity of the compound was made using [³H]QNB asligand and rat cortical membrane preparation as a source of thereceptor. The ethyl ester Compound (w) bound to the muscarinic receptors(mainly M₁ in this preparation) with high affinity (Table 9) althoughthis affinity was several-fold lower than those of the referencecompounds. The steep Hill slope close to unity indicates theantagonistic nature of its action. Compound (aa) can be evaluatedsimilarly.

TABLE 9 Affinities of tiotropium ethyl ester derivative [Compound (w)]and reference compounds for muscarinic receptors K_(i) Number Compounds(nM) Hill slope of exps. Atropine 1.9 ± 0.2 −1.10 ± 0.04 4Glycopyrrolate  0.8 ± 0.10 −1.07 ± 0.03 4 Compound (w) 7.2 ± 0.5 −1.00 ±0.04 4 The K_(i) values are for inhibition of [³H]QNB binding to ratbrain cortex membranes. Values are the mean ± S.E.M. of at least threeexperiments run in duplicate.2: Experiments with Isolated Organs

In isolated organ experiments the measurement of antimuscarinic effectof Compound (w) was carried out using guinea pig tracheal ringpreparations, where smooth muscle contraction is mediated primarily bymuscarinic M₃ cholinoceptors although activation of M₂-receptors alsoplays a role in the developing contraction. Compound (w) showedexcellent activity in this test; it was more active than atropine andonly slightly less effective than ipratropium bromide (Table 10).Compound (aa) can be tested similarly.

TABLE 10 Schild-plot analysis of the antagonism against carbachol inisolated tracheal rings of guinea pigs. Antagonist pA₂ Slope ± S.E.Atropine 8.85 0.98 ± 0.02^(a) ipratropium Br 9.18 1.11 ± 0.14^(a)Compound (w) 8.82 1.03 ± 0.08^(a) Data are presented of mean estimatesin tissue from four animals pA₂: the abscissa intercept of theSchild-plot drawn ^(a)Indicates slope estimates not significantlydifferent (P > 0.05) from unity.3. Determination of the Antagonistic Effect of Anticholinergic Agents onCharbachol Induced Bradycardia in Anesthetized Rats

Intravenous administration of the cholinomimetic carbachol causes sinusbradycardia (increasing the PP cycle and RR cycle length of the ECG) inanesthetized rats. This effect, which is mediated mainly by muscarinicM₂ receptors, can be prevented by prior administration ofanticholinergic agent. The bradycardia protective effect of Compound (w)was compared to those of atropine and ipratropium bromide in thissystem. Compound (w) was less active than an equimolar dose of atropine,and was slightly less active than a 10-fold lower dose of ipratropiumbromide indicating that Compound (w) may have lower affinity for M₂ thanM₁ or M₃ muscarinic receptors. Compound (aa) can be similarly evaluated.

Examination of the Time Course of the Anticholinergic Effect of Compound(w) and Compound (aa) in Electrically Stimulated Guinea Pig Trachea

Experimental Procedures:

The procedure described by Takahashi T. et al., (Am J Respir Crit CareMed, 150:1640-1645, 1994) was used with slight modifications.

Male Dunkin-Hartley guinea pigs (300-500 g) were exterminated; thetracheas were rapidly removed, and placed in oxygenated normal Krebsbuffer solution. The epithelium was removed and the trachea was spirallycut into 15 mm long strips. Two strips from one animal were prepared andsuspended between parallel stainless steel wire field electrodes in10-ml organ baths containing buffer solution, which was continuallygassed by a 95% 0₂ and 5% CO₂ mixture. The tissues were allowed toequilibrate for 1 h with frequent washing, under a resting tension of1.0 g.

Indomethacin 10⁻⁵(M) was present throughout the studies to block theformation of endogenous prostaglandins. Before the experiment, capsaicin(10⁻⁵M) was added and washed out 30 min after the pre-treatment todeplete endogenous tachykinins. Tissues were also pretreated withpropranolol (10⁻⁶M) 10 min before the experiment to inhibit the effectsof endogenous catecholamines.

Isometric contractile responses were measured using force-displacementtransducers (Experimetria, Hungary) connected to a Watanabe polygraph. Astimulator (CRS-ST-01-04, Experimetria) provided biphasic square-waveimpulses with a supramaximal voltage of 40 V at source and 0.5 msduration. Stimulations were applied at a frequency of 4 Hz for 15 secfollowed by a 100 sec resting interval. After at least four stableresponses of equal magnitude were obtained, the antagonist (submaximaldose) was introduced and was left in the system until the maximal effectof the drug was observed. Thereafter the test drug was washed out.Further stimulations were delivered for at least 6 additional hours oruntil the responses returned to about 50% of the original responses.Appropriate time controls were run in parallel for all studies.

Statistical Analysis

Contractile responses were expressed as the percentage of the ownmaximal contraction. The time for offset t_(1/5) or t_(1/2) of actionwas defined as the time from washout of the test antagonist toattainment of 20 or 50% recovery of cholinergic responses.

Results:

Typical tracings were obtained during the experiments. Continuous,stable, long-lasting contraction is achieved with electricalstimulation. Upon the addition of anticholinergic agents the inhibitiondevelops with varying speed, and the inhibitory effect of the compoundslast for very different periods after the washout. In FIG. 8, the timecourse of action of the different anticholinergic compounds is shown;calculated results are summarized in Table 11. In FIG. 9, the timecourse of the inhibition of the examined compounds are displayed; theresults calculated from this data are summarized in Table 12.

The differences between the on and off rates of the Compounds (w) and(aa) are very notable.

TABLE 11 Time course of action of different anticholinergics inelectrically stimulated guinea pig tracheal strips Compound t_(1/5)onset (min) t_(1/2) onset (min) Atropine 2.5 4.6 Ipratropium Br 3.0 7.0Tiotropium 8.0 12 Cpd (w) 0.6 1.2 Cpd (aa) 0.9 2.0

TABLE 12 Time course of recovery from inhibition after washing out ofdifferent anticholinergics in electrically stimulated guinea pigtracheal strips Compound t_(1/5) onset (min) t_(1/2) onset (min)Atropine 10 31 Ipratropium Br 6.5 19 Tiotropium >360 >360 Cpd (w) 60 130Cpd (aa) 27 140Investigation of the Anticholinergic Action of Compounds inAcetylcholine Induced Bronchoconstriction in Anesthetized Guinea PigsExperimental Procedure

Male Hartley guinea pigs (320±120 g) (Charles River) are housed understandard conditions. Guinea pigs are anesthetized with urethane (2 g/kg,intraperitoneally), the trachea is cannulated and the animal is respiredusing a small animal respiratory pump (Harvard Apparatus LTD, Kent UK).Respiratory back pressure is measured and recorded using a rodent lungfunction recording system (MUMED, London UK). For drug administrationthe right jugular vein is cannulated. Following the surgical preparationguinea pigs are allowed to stabilize for 20 minutes. Ten minutes beforeacetylcholine administration the animals are disconnected from theventilator and either the vehicle (10 mg lactose) or different amountsof the drug (suspended in the same amount of vehicle) are administeredintratracheally. The trachea is reconnected to the ventilator andchanges in pulmonary mechanics are followed. Acetylcholine (10 μg/kg) isadministered intravenously in every 10 minutes six times.

Active compounds exhibit a protective effect on theacetylcholine-induced bronchoconstriction provoked in this test.

This test is a model for asthma, chronic obstructive pulmonary disorderand other obstructive respiratory tract disorders in which theeffectiveness of the compounds of formula (Ia) and (Ib) can be tested.

Test for Bronchodilatory Effect of Inhaled Test Compounds in Balb/c Mice

Female BALB/c mice, weight range 19-22 g, are obtained, for example fromCharles River Laboratories (Kingston, N.C.). They receive food and waterad libitum.

Compounds for aerosol administration are prepared in sterile Dulbecco'sPhosphate Buffered Saline. Mice are placed in a carousel-style, noseonly, exposure chamber and allowed to inhale aerosols for five minutes,using an ICN SPAG-2 nebulizer. This nebulizer generates a mean aerosolparticle size of 1.3 microns at a rate of approximately 0.25 ml/minute.

Ten minutes and 36 hours later, the mice are moved to whole bodyplethysmograph chambers. Bronchoconstriction is induced in the mice byadministration of an 80 mg/ml methacholine (MC) aerosol into theplethysmograph chambers for 5 minutes. The mice are allowed to inhale anaerosol containing 80 mg/ml methacholine following inhalation treatmentwith DPBS vehicle (Dulbecco's Phosphate Buffered Saline), or 80 mg/mlmethacholine following inhalation treatment with test compound. Theaverage enhanced pause (Penh, lung resistance), corresponding to airflowresistance, is determined and statistically analyzed usingKruskal-Wallis one way ANOVA. In order to determine the baseline, salineaerosol (without methacholine) is also separately administered to themice.

This procedure is a model for inhalation treatment of asthma, chronicobstructive pulmonary disorder and other obstructive respiratory tractdisorders in which the effectiveness of the compounds of formulas (Ia)and (Ib) can be tested.

Test for Frequency of Micturition in Female Sprague-Dawley Rats

Ten female Sprague-Dawley rats having a mean weight of about 245-285 gare anesthetized with urethane (1.2 g/k, sc.). A midline incision isperformed to expose the bladder and a 23G catheter is inserted into thebladder dome for the measurement of intravesical pressure. A non-stoptransvesical cystometrogram, as described in J. Pharmacological.Methods, 15, pp. 157-167 (1986), is used, at a filling rate of 0.216ml/min. of saline, to access the filling and voiding characteristics ofthe bladder. Through the continuous cystometry method thus afforded,consecutive micturition can be recorded. Test compound is given atintravenous doses after the initial baseline micturition sequence isreliably measured for approximately 12 min. From these recordings, theabsolute values in maximum pressure obtained and the frequency ofmicturition is measured. A dose response curve illustrating the effectof test compound on the absolute micturition pressures in the range of1-50 mg/kg can be obtained. This procedure is a model for overactivebladder (OAB) in which the compounds of formula (Ia) and (Ib) can betested.

The following Examples illustrate numerous formulations suitable foradministering the compounds of formula (Ia) and (Ib) to treat variousconditions responsive to treatment with an anticholinergic agent.

In these Examples, percentages are by weight unless otherwise indicated.

EXAMPLES OF PHARMACEUTICAL FORMULATIONS Example 1

Tablets per tablet Compound of formula (Ia) or (Ib), e.g. 400 mgCompound (cc) or (ee) or (gg) or (aa) lactose 140 mg corn starch 240 mgpolyvinylpyrrolidone  15 mg magnesium stearate  5 mg 800 mg

The finely ground active substance, lactose and some of the corn starchare mixed together. The mixture is screened, then moistened with asolution of polyvinylpyrrolidone in water, kneaded, wet-granulated anddried. The granules, the remaining corn starch and the magnesiumstearate are screened and mixed together. The mixture is compressed toproduce tablets of suitable shape and size.

Example 2

Tablets per tablet Compound of formula (Ia) or (Ib), e.g. 320 mgCompound (cc) or (ee) or (gg) or (aa) lactose  55 mg corn starch 190 mgmicrocrystalline cellulose  35 mg polyvinylpyrrolidone  15 mgsodium-caroxymethyl starch  23 mg magnesium stearate  2 mg 640 mg

The finely ground active substance, some of the corn starch, lactose,microcrystalline cellulose and polyvinylpyrrolidone are mixed together,the mixture is screened and worked with the remaining corn starch andwater to form a granulate which is dried and screened. The sodiumcarboxymethyl starch and the magnesium stearate are added and mixed inand the mixture is compressed to form tablets of a suitable size.

Example 3

Ampule solution Compound of formula (Ia) or (Ib), e.g. 200 mg Compound(cc) or (ee) or (gg) or (aa) sodium chloride 50 mg water for inj. 5 ml

The active substance is dissolved in water at its own pH or optionallyat pH 5.5 to 6.5 and sodium chloride is added to make it isotonic. Thesolution obtained is filtered free from pyrogens and the filtrate istransferred under aseptic conditions into ampules which are thensterilized and sealed by fusion. The ampules contain 20 mg, 100 mg and200 mg of active substance.

Example 4

Metering aerosol Compound of formula (Ia) or (Ib), e.g. 0.020 Compound(cc) or (ee) or (gg) or (aa) Sorbitan trioleate 0.1Monofluorotrichloromethane and 2:3 ad 100 difluorodichioromethane

The suspension is transferred into a conventional aerosol container witha metering valve. Preferably, 50 μl of suspension are delivered perspray. The active substance may also be metered in higher doses ifdesired (e.g. 0.08% by weight).

Example 5

Solutions (in mg/100 ml) Compound of formula (Ia) or (Ib), e.g. 1333.3mg Compound (cc) or (ee) or (gg) or (aa) Formoterol fumarate  333.3 mgBenzalkonium chloride  10.0 mg EDTA  50.0 mg HCl(ln) ad pH 3.4

This solution may be prepared in the usual manner.

Example 6

Powder for inhalation Compound of formula (Ia) or (Ib), e.g. 24 μgCompound (cc) or (ee) or (gg) or (aa) Formoterol fumarate 6 μg Lactosemonohydrate ad 25 mg

The powder for inhalation is produced in the usual way by mixing theindividual ingredients together.

Example 7

Powder for inhalation Compound of formula (Ia) or (Ib), e.g. 40 μgCompound (cc) or (ee) or (gg) or (aa) Lactose monohydrate ad 5 mg

The powder for inhalation is produced in the usual way by mixing theindividual ingredients together.

Further Formulations Obtained Analogously to Methods Known in the Art

A: Inhalable Powders

Example 8

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) budesonide  200 lactose 4400 Total5000

Example 9

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) fluticasone propionate  125lactose 4475 Total 5000

Example 10

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) mometasone furoate × H₂O  250lactose 4350 Total 5000

Example 11

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) ciclesonide  250 lactose 4350Total 5000

Example 12

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  200Compound (cc) or (ee) or (gg) or (aa) budesonide  125 lactose 4675 Total5000

Example 13

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  200Compound (cc) or (ee) or (gg) or (aa) fluticasone propionate  200lactose 4600 Total 5000

Example 14

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  300Compound (cc) or (ee) or (gg) or (aa) mometasone furoate × H₂O  250lactose 4450 Total 5000

Example 15

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  300Compound (cc) or (ee) or (gg) or (aa) ciclesonide  250 lactose 4450Total 5000

Example 16

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) ST-126  250 lactose 4350 Total5000

Example 17

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  200Compound (cc) or (ee) or (gg) or (aa) ST-126  125 lactose 4675 Total5000

Example 18

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate  200 lactose4400 Total 5000

Example 19

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) etiprednol dichloroacetate  200lactose 4400 Total 5000

Example 20

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate  125 lactose4475 Total 5000

Example 21

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  200Compound (cc) or (ee) or (gg) or (aa) etiprednol dichloroacetate  125lactose 4675 Total 5000

Example 22

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate  200 Δ¹ -cortienic acid methyl ester  200 lactose 4200 Total 5000

Example 23

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate  200 Δ¹ -cortienic acid  200 lactose 4200 Total 5000

Example 24

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  400Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate  125 Δ¹ -cortienic acid or  125 Δ¹ - cortienic acid methyl ester lactose 4350Total 5000

Example 25

μg Ingredients per capsule Compound of formula (Ia) or (Ib), e.g.  200Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate  125 Δ¹ -cortienic acid or  125 Δ¹ - cortienic acid methyl ester lactose 4550Total 5000B. Propellant-Containing Aerosols for Inhalation (wherein TG 134a is1,1,1,2-tetrafluoroethane and TG 227 is1,1,1,2,3,3,3-heptafluoropropane)

Example 26 Suspension Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.2Compound (cc) or (ee) or (gg) or (aa) budesonide 0.4 soya lecithin 0.2TG134a:TG227 (2:3) to 100

Example 27 Suspension Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.08Compound (cc) or (ee) or (gg) or (aa) fluticasone propionate 0.3isopropyl myristate 0.1 TG227 to 100

Example 28 Suspension Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.08Compound (cc) or (ee) or (gg) or (aa) mometasone furoate × H₂O 0.6isopropyl myristate 0.1 TG227 to 100

Example 29 Suspension Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.08Compound (cc) or (ee) or (gg) or (aa) ciclesonide 0.4 isopropylmyristate 0.1 TG134a:TG227 (2:3) to 100

Example 30 Suspension Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.17Compound (cc) or (ee) or (gg) or (aa) ciclesonide 0.4 absolute ethanol0.5 isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 31 Solution Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.17Compound (cc) or (ee) or (gg) or (aa) fluticasone propionate 0.2absolute ethanol 0.5 isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 32 Solution Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.17Compound (cc) or (ee) or (gg) or (aa) mometasone furoate × H₂O 0.6absolute ethanol 0.5 isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 33 Solution Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.17Compound (cc) or (ee) or (gg) or (aa) ciclesonide 0.4 absolute ethanol0.5 isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 34 Solution Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.17Compound (cc) or (ee) or (gg) or (aa) ST-126 0.6 absolute ethanol 0.5isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 35 Solution Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.17Compound (cc) or (ee) or (gg) or (aa) ST-126 0.4 absolute ethanol 0.5isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 36

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.2Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate 0.4 soyalecithin 0.2 TG134a:TG227 (2:3) to 100

Example 37

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.08Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate 0.3isopropyl myristate 0.1 TG227 to 100

Example 38

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.08Compound (cc) or (ee) or (gg) or (aa) etiprednol dichlorocetate 0.4isopropyl myristate 0.1 TG227 to 100

Example 39

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.08Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate 0.4isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 40

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.17Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate 0.4 absoluteethanol 0.5 isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 41

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.2Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate 0.4 Δ¹ -cortienic acid or Δ¹ - cortienic acid methyl ester 0.4 soya lecithin 0.2TG134a:TG227 (2:3) to 100

Example 42

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.08Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate 0.3 Δ¹ -cortienic acid or Δ¹ - cortienic acid methyl ester 0.3 isopropylmyristate 0.1 TG227 to 100

Example 43

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.16Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate 0.4 Δ¹ -cortienic acid or Δ¹ - cortienic acid 0.4 methyl ester isopropylmyristate 0.1 TG227 to 100

Example 44

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.08Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate 0.4 Δ¹ -cortienic acid or Δ¹ - cortienic acid 0.4 methyl ester isopropylmyristate 0.1 TG134a:TG227 (2:3) to 100

Example 45

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g.  0.17Compound (cc) or (ee) or (gg) or (aa) loteprednol etabonate 0.4 Δ¹ -cortienic acid or Δ¹ - cortienic acid 0.4 methyl ester absolute ethanol0.5 isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100C. Ophthalmic Formulations

Example 46

EYE DROPS Compound of formula (Ia) or (Ib), e.g. 0.20% w/v Compound (cc)or (ee) or (gg) or (aa) Tween 80  2.5% w/v Ethanol 0.75% w/vBenzalkonium chloride 0.02% w/v Phenyl ethanol 0.25% w/v Sodium chloride0.60% w/v Water for injection q.s. 100 volumes

Example 47

EYE DROPS Compound of formula (Ia) or (Ib), e.g. 0.16% w/v Compound (cc)or (ee) or (gg) or (aa) Tween 80  2.5% w/v Ethanol 0.75% w/vBenzalkonium chloride 0.02% w/v Phenyl ethanol 0.25% w/v Sodium chloride0.60% w/v Water for injection q.s. 100 volumes

Example 48

EYE DROPS Compound of formula (Ia) or (Ib), e.g. 0.14% w/v Compound (cc)or (ee) or (gg) or (aa) Povidone 0.6% w/v Benzalkonium chloride 0.02%w/v Sodium edetate U.S.P. 0.10% w/v Glycerin U.S.P. 2.5% w/v TyloxapolU.S.P. 3.0% w/v Sodium chloride 0.3% w/v Sodium γ-aminobutyrate 1.0% w/vSterile distilled water q.s. 100 volumes

The ingredients listed above are combined, then the pH is checked and,if necessary, adjusted to 5.0-5.5 by basifying with sodium hydroxide oracidifying with hydrochloric acid.

Yet other compositions of the invention can be conveniently formulatedusing known techniques.

While this description has been couched in terms of various preferred orexemplary embodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the foregoing be limited only by the broadest statements hereinand by the scope of the following claims, including equivalents thereof.

1. The compound having the formula:


2. A pharmaceutical composition comprising an anticholinergicallyeffective amount of the compound as claimed in claim 1 and a non-toxicpharmaceutically acceptable carrier therefor.
 3. A pharmaceuticalcombination comprising the compound as claimed in claim 1 and anantiinflammatory corticosteroid, betamimetic agent or antiallergicagent, in a combined amount effective for reducing or inhibiting thedevelopment of, or alleviating the symptoms of, chronic obstructivepulmonary disease or asthma.
 4. A pharmaceutical combination accordingto claim 3, wherein the antiinflammatory corticosteroid is loteprednoletabonate or etiprednol dichloroacetate.
 5. A pharmaceutical combinationaccording to claim 4, wherein the antiinflammatory corticosteroid isloteprednol etabonate.
 6. A pharmaceutical combination according toclaim 5, further comprising an enhancing agent for the loteprednoletabonate selected from the group consisting of: (a)11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylic acid; (b)11β,17α-dihydroxyandrost-1,4-dien-3-one-17β-carboxylic acid; (c) methyl11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylate; (d) ethyl11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylate; (e) methyl11β,17α-dihydroxyandrost-1,4-dien-3-one-17β-carboxylate; and (f) ethyl11β,17α-dihydroxyandrost-1,4-dien-3-one-17β-carboxylate.